Alveolar Epithelial Apoptosis Research Articles

Gut microbiota-derived succinate aggravates acute lung injury after intestinal ischaemia/reperfusion in mice.

Acute lung injury (ALI) is a major cause of morbidity and mortality after intestinal ischaemia/reperfusion (I/R). The gut microbiota and its metabolic byproducts act as important modulators of the gut-lung axis. This study aimed to define the role of succinate, a key microbiota metabolite, in intestinal I/R-induced ALI progression. Gut and lung microbiota of mice subjected to intestinal I/R were analysed using 16S rRNA gene sequencing. Succinate level alterations were measured in germ-free mice or conventional mice treated with antibiotics. Succinate-induced alveolar macrophage polarisation and its effects on alveolar epithelial apoptosis were evaluated in succinate receptor 1 (Sucnr1)-deficient mice and in murine alveolar macrophages transfected with Sucnr1-short interfering RNA. Succinate levels were measured in patients undergoing cardiopulmonary bypass, including intestinal I/R. Succinate accumulated in lungs after intestinal I/R, and this was associated with an imbalance of succinate-producing and succinate-consuming bacteria in the gut, but not the lungs. Succinate accumulation was absent in germ-free mice and was reversed by gut microbiota depletion with antibiotics, indicating that the gut microbiota is a source of lung succinate. Moreover, succinate promoted alveolar macrophage polarisation, alveolar epithelial apoptosis and lung injury during intestinal I/R. Conversely, knockdown of Sucnr1 or blockage of SUCNR1 in vitro and in vivo reversed the effects of succinate by modulating the phosphoinositide 3-kinase-AKT/hypoxia-inducible factor-1α pathway. Plasma succinate levels significantly correlated with intestinal I/R-related lung injury after cardiopulmonary bypass. Gut microbiota-derived succinate exacerbates intestinal I/R-induced ALI through SUCNR1-dependent alveolar macrophage polarisation, identifying succinate as a novel target for gut-derived ALI in critically ill patients.

Sensitization of GSH synthesis by curcumin curtails acrolein-induced alveolar epithelial apoptosis via Keap1 cysteine conjugation: A randomized controlled trial and experimental animal model of pneumonitis

IntroductionEpidemiological studies have reported an association between exposures to ambient air pollution and respiratory diseases, including chronic obstructive pulmonary disease (COPD). Pneumonitis is a critical driving factor of COPD and exposure to air pollutants (e.g., acrolein) is associated with increased incidence of pneumonitis. ObjectivesCurrently available anti-inflammatory therapies provide little benefit against respiratory diseases. To this end, we investigated the preventive role of curcumin against air pollutant-associated pneumonitis and its underlying mechanism. MethodsA total of 40 subjects was recruited from Chengdu, China which is among the top three cities in terms of respiratory mortality related to air pollution. The participants were randomly provided either placebo or curcumin supplements for 2 weeks and blood samples were collected at the baseline and at the end of the intervention to monitor systemic markers. In our follow up mechanistic study, C57BL/6 mice (n = 40) were randomly allocated into 4 groups: Control group (saline + no acrolein), Curcumin only group (curcumin + no acrolein), Acrolein only group (saline + acrolein), and Acrolein + Curcumin group (curcumin + acrolein). Curcumin was orally administered at 100 mg/kg body weight once a day for 10 days, and then the mice were subjected to nasal instillation of acrolein (5 mg/kg body weight). Twelve hours after single acrolein exposure, all mice were euthanized. ResultsCurcumin supplementation, with no noticeable adverse responses, reduced circulating pro-inflammatory cytokines in association with clinical pneumonitis as positive predictive while improving those of anti-inflammatory cytokines. In the pre-clinical study, curcumin reduced pneumonitis manifestations by suppression of intrinsic and extrinsic apoptotic signaling, which is attributed to enhanced redox sensing of Nrf2 and thus sensitized synthesis and restoration of GSH, at least in part, through curcumin-Keap1 conjugation. ConclusionsOur study collectively suggests that curcumin could provide an effective preventive measure against air pollutant-enhanced pneumonitis and thus COPD.

PACS2\u2013TRPV1 axis is required for ER\u2013mitochondrial tethering during ER stress and lung fibrosis

Endoplasmic reticulum (ER) and mitochondria (mito) play a vital role in alveolar type II cell (AEC2) homeostasis and are both stressed in patients with idiopathic pulmonary fibrosis (IPF). Up to now, no data are available with regard to ER–mito cross talk and tethering under conditions of IPF. We here demonstrate that ER–mitochondrial tethering is reduced upon experimental ER stress in vitro and in the IPF AECII ex vivo, and this is—at least in part—due to decreased phosphofurin acidic cluster sorting protein 2 (PACS-2, also called PACS2) protein levels. PACS2 levels are influenced by its interaction with the transient receptor potential cation channel subfamily V member 1 (TRPV1) and can be experimentally modified by the TRPV1-modulating drug capsaicin (CPS). Employing alveolar epithelial cells with overexpression of the terminal ER stress signaling factor Chop or the IPF-associated surfactant protein C mutation (SPCΔexon4) in vitro, we observed a restoration of PACS2 levels upon treatment with CPS. Similarly, treatment of precision cut lung slices from IPF patients with CPS ex vivo forwarded similar effects. Importantly, in all models such kind of intervention also greatly reduced the extent of alveolar epithelial apoptosis. We therefore conclude that therapeutic targeting of the PACS2–TRPV1 axis represents an interesting novel, epithelial-protective approach in IPF.

Role of phospholipase D in bleomycin-induced mitochondrial reactive oxygen species generation, mitochondrial DNA damage, and pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a pernicious lung disease characterized by alveolar epithelial apoptosis, dysregulated repair of epithelial injury, scar formation, and respiratory failure. In this study, we identified phospholipase D (PLD)-generated phosphatidic acid (PA) signaling in the development of pulmonary fibrosis (PF). Of the PLD isoenzymes, the protein expression of PLD2, but not PLD1, was upregulated in lung tissues from IPF patients and bleomycin challenged mice. Both PLD1 (Pld1-/-)- and PLD2 (Pld2-/-)-deficient mice were protected against bleomycin-induced lung inflammation and fibrosis, thereby establishing the role of PLD in fibrogenesis. The role of PLD1 and PLD2 in bleomycin-induced lung epithelial injury was investigated by infecting bronchial airway epithelial cells (Beas2B) with catalytically inactive mutants of PLD (hPLD1-K898R or mPld2-K758R) or downregulation of expression of PLD1 or PLD2 with siRNA. Bleomycin stimulated mitochondrial (mt) superoxide production, mtDNA damage, and apoptosis in Beas2B cells, which was attenuated by the catalytically inactive mutants of PLD or PLD2 siRNA. These results show a role for PLD1 and PLD2 in bleomycin-induced generation of mt reactive oxygen species, mt DNA damage, and apoptosis of lung epithelial cells in mice. Thus, PLD may be a novel therapeutic target in ameliorating experimental PF in mice.

Decreased Serum Levels of Irisin are Associated with the Development of Chronic Lung Allograft Dysfunction after Bilateral Living-Donor Lobar Lung Transplantation

Purpose Irisin is a skeletal muscle cell-derived myokine associated with physical activity. Recently, decreased serum irisin levels have been shown to cause alveolar epithelial apoptosis resulting in lung emphysema in patients with chronic obstructive pulmonary disease. Similar to the patients with emphysema, the patients with chronic lung allograft dysfunction (CLAD) after living-donor lobar lung transplantation (LDLLT) could show emphysematous changes, because in LDLLT, small lobar grafts expand to fit into the recipient's chest cavity due to the size mismatch. However, the relationship between serum irisin levels and CLAD after LDLLT remains unknown. Methods Blood samples were collected from a total of 33 patients who underwent bilateral LDLLT, including patients with CLAD (the CLAD group, n=11) and those without CLAD (the non-CLAD group, n=22). Serum samples were assayed for irisin using ELISA kit and compared between the two groups. Appropriate cut-off values of irisin level were set for the diagnosis of CLAD. Results The median follow-up period was 4366 (1055-7180) days. Serum irisin levels of the CLAD group were significantly lower than those of the non-CLAD group (9.5 ± 3.4 vs. 13.2 ± 4.2 ng/mL, P = 0.018) (Fig. 1). An ROC analysis of the performance of the serum irisin level as a marker of CLAD yielded an AUC of 0.76 (sensitivity=0.91 and specificity=0.64 at a threshold level of 12.6 ng/mL). Moreover, patients with irisin level ≥12.6 ng/mL (n=16) showed significantly better CLAD-free survival than those with irisin level Conclusion Decreased serum irisin levels are associated with the development of CLAD after bilateral LDLLT. Irisin might be a novel biomarker for the diagnosis of CLAD after LDLLT.

Nrf2 transfection enhances the efficacy of human amniotic mesenchymal stem cells to repair lung injury induced by lipopolysaccharide.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are clinical emergencies with no effective pharmaceutical treatment. This study aims to determine the protective effects of Nrf2-transfected human amniotic mesenchymal stem cells (hAMSCs) against lipopolysaccharide (LPS)-induced lung injury in mice. hAMSCs stably transfected with Nrf2 or green fluorescent protein control were transplanted into male C57BL/6 mice via the tail vein 4 h after intratracheal instillation of LPS. At 3, 7, and 14 days after cell transplantation, total lung injury score (the Smith score) was determined by hematoxylin and eosin staining. Lung fibrosis was assessed by Masson's trichrome staining. Alveolar epithelial apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick end labeling staining. The plasma levels of interleukin (IL)-1β, IL-6, and IL-10 were determined by enzyme-linked immunosorbent assays (ELISA). The homing and differentiation of hAMSCs into type II alveolar epithelial (AT II) cells were examined by immunofluorescent staining and/or western blot analysis. Nrf2, mRNA, and protein expression in lungs were examined by qRT-PCR and western blot analysis, and DNA-binding activity of Nrf2 was detected by ELISA. We found that, compared with control hAMSCs, treatment with Nrf2-overexpressing hAMSCs led to further reduced lung injury, lung fibrosis, and inflammation in LPS-challenged mice. Nrf2-overexpressing hAMSCs also exhibited increased cell retention in the lung, more efficient differentiation into AT II cells, and more prominent effects on the increased mRNA and protein expression as well as DNA-binding activity of Nrf2 than control. These results support Nrf2-overexpressing hAMSCs as a potential cell-based therapy for clinical ALI/ARDS.

Effects of lipoxin A4 on human type II alveolar epithelial cell wound repair, proliferation and apoptosis

Objective To evaluate the effects of lipoxin A4 (LXA4) on human type Ⅱ alveolar epithelial cell wound repair, proliferation and apoptosis. Methods Experiment Ⅰ Human type Ⅱ alveolar epithelial cells were inoculated in 24-well plates and divided into 4 groups (n=10 each) using a random number table: control group (group C), 1 nmol/L LXA4 group (group L1), 10 nmol/L LXA4 group (group L2) and 100 nmol/L LXA4 group (group L3). Cells were cultured in normal culture atmosphere in group C. Cells were incubated with 1, 10 and 100 nmol/L LXA4 in L1, L2 and L3 groups, respectively.The scratch wound assay was performed at 36 h of culture or incubation.Cell proliferation was measured at 24 h of culture or incubation.Experiment Ⅱ Human type Ⅱ alveolar epithelial cells were inoculated in 96-well plates and divided into 5 groups using a random number table: control group (group C, n=10), Fas-ligand group(n=10), Fas-ligand+ LXA4 group(n=10), Fas-ligand+ TNF-α group(n=5) and Fas-ligand+ TNF-α+ LXA4 group(n=5). Cells were incubated with 100 ng/ml Fas-Ligand, 100 ng/ml Fas-Ligand plus 100 nmol/L LXA4, 100 ng/ml Fas-Ligand plus 100 ng/ml TNF-α, and 100 ng/ml Fas-Ligand plus 100 ng/ml TNF-α plus 100 nmol/L LXA4 in Fas-ligand, Fas-ligand+ LXA4, Fas-ligand+ TNF-α, and Fas-ligand+ TNF-α+ LXA4 groups, respectively.The cell viability was measured at 24 h of culture or incubation.Cell apoptosis was detected using the flow cytometry, and apoptosis rate was calculated in C, Fas-ligand and Fas-ligand+ LXA4 groups. Results Experiment Ⅰ Compared with group C, the percentage of cell repair size and percentage of proliferation were significantly increased in L1, L2 and L3 groups (P 0.05). Experiment Ⅱ Compared with group C, the cell viability was significantly decreased, and the apoptosis rate was increased in group Fas-ligand, the cell viability was significantly decreased in group Fas-ligand+ TNF-α (P 0.05). Compared with group Fas-ligand, the cell viability was significantly increased, and the apoptosis rate was decreased in group Fas-ligand+ LXA4 (P<0.05). The cell viability was significantly higher in group Fas-ligand+ TNF-α+ LXA4 than in group Fas-ligand+ TNF-α (P<0.01). Conclusion LXA4 can promote human type Ⅱ alveolar epithelial cell wound repair and proliferation and inhibit the apoptosis. Key words: Lipoxins; Pulmonary alveoli; Epithelial cells; Cell proliferation; Apoptosis; Recovery of function

Nintedanib reduces ventilation-augmented bleomycin-induced epithelial-mesenchymal transition and lung fibrosis through suppression of the Src pathway.

Mechanical ventilation (MV) used in patients with acute respiratory distress syndrome (ARDS) can increase lung inflammation and pulmonary fibrogenesis. Src is crucial in mediating the transforming growth factor (TGF)‐β1‐induced epithelial–mesenchymal transition (EMT) during the fibroproliferative phase of ARDS. Nintedanib, a multitargeted tyrosine kinase inhibitor that directly blocks Src, has been approved for the treatment of idiopathic pulmonary fibrosis. The mechanisms regulating interactions among MV, EMT and Src remain unclear. In this study, we suggested hypothesized that nintedanib can suppress MV‐augmented bleomycin‐induced EMT and pulmonary fibrosis by inhibiting the Src pathway. Five days after administrating bleomycin to mimic acute lung injury (ALI), C57BL/6 mice, either wild‐type or Src‐deficient were exposed to low tidal volume (VT) (6 ml/kg) or high VT (30 ml/kg) MV with room air for 5 hrs. Oral nintedanib was administered once daily in doses of 30, 60 and 100 mg/kg for 5 days before MV. Non‐ventilated mice were used as control groups. Following bleomycin exposure in wild‐type mice, high VT MV induced substantial increases in microvascular permeability, TGF‐β1, malondialdehyde, Masson's trichrome staining, collagen 1a1 gene expression, EMT (identified by colocalization of increased staining of α‐smooth muscle actin and decreased staining of E‐cadherin) and alveolar epithelial apoptosis (P < 0.05). Oral nintedanib, which simulated genetic downregulation of Src signalling using Src‐deficient mice, dampened the MV‐augmented profibrotic mediators, EMT profile, epithelial apoptotic cell death and pathologic fibrotic scores (P < 0.05). Our data indicate that nintedanib reduces high VT MV‐augmented EMT and pulmonary fibrosis after bleomycin‐induced ALI, partly by inhibiting the Src pathway.

ABSTRACT 485

Background and aims: The production of reactive oxygen species (ROS) during hyperoxia is thought to contribute to alveolar epithelial apoptosis. Aims: To explore the molecular mechanisms of oxidative stress-induced alveolar epithelial cell apoptosis and assess the possible cytoprotective effects of N-acetylcysteine (NAC). Methods: 500 μM H2O2 treatment induced primary alveolar type II epithelial cells apoptosis, during this course a rapid increase of phosphorylation levels of JNK activated by H2O2 was found. The apoptosis of ATII cells can be prevented by SP600125, a specific inhibitor of JNK. SP600125 also attenuated Bax expression and p53 nuclear accumulation induced by H2O2. Results: Pretreatment of ATII cells with NAC (5 mM) 0.5 h before H2O2 would markedly decreased cell apoptosis and the activation of JNK. The high level of intracellular ROS induced by H2O2 was also significantly attenuated by NAC pretreatment. Conclusions: Taken together, these findings suggest JNK is the upstream of Bax and p53 in ATII cells apoptosis induced by oxidative stress, and NAC attenuated cells apoptosis through suppressing intracellular ROS generation and JNK activation.

Antioxidant defense of peroxiredoxin 6 in lung: recent progress

Alveolar epithelial apoptosis and injury induced by oxidative stress in pulmonary diseases have drawn great attention from researchers.Peroxiredoxin 6(Prdx6),endowed with the activities of both glutathione(GSH)peroxidase and phospholipase A2(PLA2),can effectively fight against oxidative stress and alleviate oxidative stress-induced lung injury,and therefore plays an important role in pulmonary diseases.This review focuses on the structure,function,distribution and expression regulation of Prdx6,and summarizes the role of Prdx6 in pulmonary antioxidant defense and its regulatory mechanism based on experimental studies.

18:1/18:1-Dioleoyl-phosphatidylglycerol prevents alveolar epithelial apoptosis and profibrotic stimulus in a neonatal piglet model of acute respiratory distress syndrome

Background18:1/18:1-Dioleoyl-phosphatidylgycerol (DOPG) is a surfactant phospholipid that is nearly non-detectable in neonatal surfactant films. When alveolar macrophages are exposed to DOPG in vitro, secretory phospholipase A2 (sPLA2) production is blocked, resulting in suppressed macrophage activity and improved surfactant function. We investigated whether the addition of DOPG to a commercially available surfactant preparation would improve lung function in a neonatal piglet model of acute respiratory distress syndrome. Materials and methodsRespiratory failure was achieved by triple-hit lung injury (repeated broncho-alveolar lavage, injurious ventilation, tracheal lipopolysaccharide instillation, each intervention 24 h apart) in twenty-four domestic piglets aged 2–6 days and subject to mechanical ventilation. Following each lung injury protocol the piglets were treated with surfactant alone or with surfactant + DOPG. ResultsWithin 72 h of mechanical ventilation, we observed significantly improved gas exchange (oxygenation and ventilation), lung mechanics (compliance and resistance of the respiratory system), and pulmonary oedema (extra-vascular lung water index) in the surfactant + DOPG group. This favourable clinical effect could be attributed to improved surfactant function, reduced sPLA2 secretion, inhibition of macrophage migration, reduced alveolar epithelial apoptosis, and suppression of amphiregulin and TGF-β1 expression in pulmonary tissues as a prerequisite for fibrous lung repair. ConclusionsWe conclude that surfactant fortified by DOPG preserves lung function, and prevents alveolar epithelial injury and fibrous stimulus by reduction of sPLA2 in a neonatal model of acute respiratory distress syndrome without any relevant discernable side effects. Hence, DOPG supplementation in a neonatal lung exerts important function protecting effects and seems to be justified in cases of overwhelming pulmonary inflammation.

Topical application of phosphatidyl‐inositol‐3,5‐bisphosphate for acute lung injury in neonatal swine

Hypoxemic respiratory failure of the neonatal organism involves increased acid sphingomyelinase (aSMase) activity and production of ceramide, a second messenger of a pro-inflammatory pathway that promotes increased vascular permeability, surfactant alterations and alveolar epithelial apoptosis. We comparatively assessed the benefits of topical aSMase inhibition by either imipramine (Imi) or phosphatidylinositol-3,5-bisphosphate (PIP2) when administered into the airways together with surfactant (S) for fortification. In this translational study, a triple-hit acute lung injury model was used that entails repeated airway lavage, injurious ventilation and tracheal lipopolysaccharide instillation in newborn piglets subject to mechanical ventilation for 72 hrs. After randomization, we administered an air bolus (control), S, S+Imi, or S+PIP2. Only in the latter two groups we observed significantly improved oxygenation and ventilation, dynamic compliance and pulmonary oedema. S+Imi caused systemic aSMase suppression and ceramide reduction, whereas the S+PIP2 effect remained compartmentalized in the airways because of the molecule's bulky structure. The surfactant surface tensions improved by S+Imi and S+PIP2 interventions, but only to a minor extent by S alone. S+PIP2 inhibited the migration of monocyte-derived macrophages and granulocytes into airways by the reduction of CD14/CD18 expression on cell membranes and the expression of epidermal growth factors (amphiregulin and TGF-β1) and interleukin-6 as pro-fibrotic factors. Finally we observed reduced alveolar epithelial apoptosis, which was most apparent in S+PIP2 lungs. Exogenous surfactant “fortified” by PIP2, a naturally occurring surfactant component, improves lung function by topical suppression of aSMase, providing a potential treatment concept for neonates with hypoxemic respiratory failure.

The Alveolar Epithelium Determines Susceptibility to Lung Fibrosis in Hermansky-Pudlak Syndrome

Hermansky-Pudlak syndrome (HPS) is a family of recessive disorders of intracellular trafficking defects that are associated with highly penetrant pulmonary fibrosis. Naturally occurring HPS mice reliably model important features of the human disease, including constitutive alveolar macrophage activation and susceptibility to profibrotic stimuli. To decipher which cell lineage(s) in the alveolar compartment is the predominant driver of fibrotic susceptibility in HPS. We used five different HPS and Chediak-Higashi mouse models to evaluate genotype-specific fibrotic susceptibility. To determine whether intrinsic defects in HPS alveolar macrophages cause fibrotic susceptibility, we generated bone marrow chimeras in HPS and wild-type mice. To directly test the contribution of the pulmonary epithelium, we developed a transgenic model with epithelial-specific correction of the HPS2 defect in an HPS mouse model. Bone marrow transplantation experiments demonstrated that both constitutive alveolar macrophage activation and increased susceptibility to bleomycin-induced fibrosis were conferred by the genotype of the lung epithelium, rather than that of the bone marrow-derived, cellular compartment. Furthermore, transgenic epithelial-specific correction of the HPS defect significantly attenuated bleomycin-induced alveolar epithelial apoptosis, fibrotic susceptibility, and macrophage activation. Type II cell apoptosis was genotype specific, caspase dependent, and correlated with the degree of fibrotic susceptibility. We conclude that pulmonary fibrosis in naturally occurring HPS mice is driven by intracellular trafficking defects that lower the threshold for pulmonary epithelial apoptosis. Our findings demonstrate a pivotal role for the alveolar epithelium in the maintenance of alveolar homeostasis and regulation of alveolar macrophage activation.

Inositol–Trisphosphate Reduces Alveolar Apoptosis and Pulmonary Edema in Neonatal Lung Injury

D-myo-inositol-1,2,6-trisphosphate (IP3) is an isomer of the naturally occurring second messenger D-myo-inositol-1,4,5-trisphosphate, and exerts anti-inflammatory and antiedematous effects in the lung. Myo-inositol (Inos) is a component of IP3, and is thought to play an important role in the prevention of neonatal pulmonary diseases such as bronchopulmonary dysplasia and neonatal acute lung injury (nALI). Inflammatory lung diseases are characterized by augmented acid sphingomyelinase (aSMase) activity leading to ceramide production, a pathway that promotes increased vascular permeability, apoptosis, and surfactant alterations. A novel, clinically relevant triple-hit model of nALI was developed, consisting of repeated airway lavage, injurious ventilation, and lipopolysaccharide instillation into the airways, every 24 hours. Thirty-five piglets were randomized to one of four treatment protocols: control (no intervention), surfactant alone, surfactant + Inos, and surfactant + IP3. After 72 hours of mechanical ventilation, lungs were excised from the thorax for subsequent analyses. Clinically, oxygenation and ventilation improved, and extravascular lung water decreased significantly with the S + IP3 intervention. In pulmonary tissue, we observed decreased aSMase activity and ceramide concentrations, decreased caspase-8 concentrations, reduced alveolar epithelial apoptosis, the reduced expression of interleukin-6, transforming growth factor-β1, and amphiregulin (an epithelial growth factor), reduced migration of blood-borne cells and particularly of CD14(+)/18(+) cells (macrophages) into the airspaces, and lower surfactant surface tensions in S + IP3-treated but not in S + Inos-treated piglets. We conclude that the admixture of IP3 to surfactant, but not of Inos, improves gas exchange and edema in our nALI model by the suppression of the governing enzyme aSMase, and that this treatment deserves clinical evaluation.

Loss of Shp2 in alveoli epithelia induces deregulated surfactant homeostasis, resulting in spontaneous pulmonary fibrosis

Type II alveolar epithelial (AT-II) cells produce pulmonary surfactant proteins that are essential for alveolar function. AT-II is a major target in lung injury, and ineffective repair of damaged alveolar epithelia has been postulated to cause pulmonary fibrosis. Previous studies have shown that tyrosine phosphatase Shp2 is expressed highly in the embryonic lung epithelial buds, and Shp2 activity is required for FGF-induced lung branching morphogenesis. To investigate in vivo function of pulmonary Shp2, we generated alveoli epithelia-specific Shp2-knockout (Shp2(Δ/Δ)) mice. Shp2(Δ/Δ) mice exhibit marked reduction in surfactant proteins, disorganized lamellar bodies, increased alveolar epithelial apoptosis, and interstitial pulmonary fibrosis without preceding inflammation. Mechanistically, Shp2 acts to mediate expression of thyroid transcription factor 1 (TTF1) and ATP-binding cassette subfamily A member 3 (ABCA3). Shp2 also plays a central role in mediating FGF/GAB/ERK activity, required for epithelial repair program. Together, our results identify a novel role of tyrosine phosphatase Shp2 in surfactant homeostasis, and deregulation of Shp2 triggers spontaneous pulmonary fibrosis with minimal inflammation.

Exudate macrophages attenuate lung injury by the release of IL-1 receptor antagonist in gram-negative pneumonia

Exudate macrophages are key players in host defense towards invading pathogens. Their anti-inflammatory and epithelial-protective potential in gram-negative pneumonia, however, remains elusive. Objective: We investigated whether exudate macrophages contributed to preservation of alveolar epithelial barrier integrity and analyzed the molecular pathways involved. Methods: We evaluated the anti-inflammatory and epithelial-protective effects of exudate macrophages in a model of LPS- and K. pneumoniae-induced lung injury comparing wild-type and CCR2 (CC-chemokine receptor 2)-deficient mice with defective lung macrophage recruitment and in in vitro studies using primary alveolar epithelial cells. CCR2–/– mice exhibited enhanced alveolar epithelial cell apoptosis and lung leakage upon intratracheal LPS treatment which could be attributed to lack of exudate macrophage recruitment from the circulating pool as demonstrated in a model of wild-type/CCR2–/– bone-marrow chimeric mice. Among various anti-inflammatory and proliferative mediators analysed, the endogenous counterpart of resident macrophage-expressed interleukin-1β (IL-1β), interleukin-1 receptor antagonist (IL-1ra), was highly upregulated in flow-sorted exudate macrophages in LPS-treated wild-type mice. LPS/IL-1β-induced impairment of alveolar epithelial cell integrity was antagonized by IL-1ra in vitro. Finally, intratracheal substitution of IL-1ra or intravenous adoptive transfer of IL-1ra+/+ but not IL-1ra–/– blood mononuclear cells attenuated alveolar inflammation, epithelial apoptosis, and loss of barrier function in LPS-challenged or K. pneumoniae-infected CCR2–/– mice and enhanced survival after K. pneumoniae infection. We conclude that recruited lung macrophages attenuate IL-1β-mediated acute lung injury in gram-negative pneumonia by release of IL-1ra.

Exudate Macrophages Attenuate Lung Injury by the Release of IL-1 Receptor Antagonist in Gram-negative Pneumonia

Exudate macrophages are key players in host defense toward invading pathogens. Their antiinflammatory and epithelial-protective potential in gram-negative pneumonia, however, remains elusive. We investigated whether exudate macrophages contributed to preservation of alveolar epithelial barrier integrity and analyzed the molecular pathways involved. We evaluated the antiinflammatory and epithelial-protective effects of exudate macrophages in a model of LPS- and Klebsiella pneumoniae-induced lung injury comparing wild-type and CC-chemokine receptor 2 (CCR2)-deficient mice with defective lung macrophage recruitment and in in vitro studies using primary alveolar epithelial cells. CCR2(-/-) mice exhibited enhanced alveolar epithelial cell apoptosis and lung leakage on intratracheal LPS treatment, which could be attributed to lack of exudate macrophage recruitment from the circulating pool as demonstrated in a model of wild-type/CCR2(-/-) bone-marrow chimeric mice. Among various antiinflammatory and proliferative mediators analyzed, the endogenous counterpart of resident macrophage-expressed IL-1β, IL-1 receptor antagonist (IL-1ra), was highly up-regulated in flow-sorted exudate macrophages in LPS-treated wild-type mice. LPS/IL-1β-induced impairment of alveolar epithelial cell integrity was antagonized by IL-1ra in vitro. Finally, intratracheal substitution of IL-1ra or intravenous adoptive transfer of IL-1ra(+/+) but not IL-1ra(-/-) blood mononuclear cells attenuated alveolar inflammation, epithelial apoptosis, and loss of barrier function in LPS-challenged or K. pneumoniae-infected CCR2(-/-) mice and enhanced survival after K. pneumoniae infection. We conclude that recruited lung macrophages attenuate IL-1β-mediated acute lung injury in gram-negative pneumonia by release of IL-1ra.

Angiotensin Receptor Type I Blockade Inhibits Apoptosis in Meconium-Instilled Rabbit Lungs

Background and objectives: In prior studies we demonstrated that meconium-induced lung cell death by apoptosis was associated with activation of the local pulmonary renin-angiotensin system (RASL). Alveolar epithelial apoptosis requires the authocrine synthesis and proteolytic processing of angiotensinogen (AGT) to Angiotensin II (ANG II). Inhibitors of angiotensin converting enzyme (ACE) block meconium-induced apoptosis. ANG II plays an essential role in vascular homeostasis and lung injury. The objectives of this study were to evaluate expression of AGT, ANG II and Caspase 3 in meconium and saline treated newborn lungs and to study mechanisms of its inhibition by a selective antagonist of the AT1 receptor. Methods: Two week old rabbits were studied. Three treatment groups were studied (six rabbit pups in each group): Group 1: rabbits instilled with saline; Group 2: rabbits instilled with 10% meconium; Group 3: Losartan pretreated followed by meconium-instillation. Three groups of A549 human lung epithelial cells were studies as well. Group 4: AGT pretreated and then meconium-exposed cells; Group 5: ANG II pretreated and then meconium exposed cells and Group 6: Caspase 3 inhibitor ZVAD-fmk pretreated and then meconium exposed cells. AGT, ANG II and Caspase 3 were evaluated and compared with and without inhibitors in meconium and control groups. Results: In Situ End Labeling (ISEL) and Caspase 3 assays showed that purified ANG II induced dose dependent apoptosis in rabbit lung lavage cells and the human A549 lung epithelial cell line. Apoptosis also was induced by purified AGT. The increase in apoptotic cells was accompanied by increases in ANG II and Caspase 3 activities. In both airway epithelium and alveolar wall cells, measures of apoptosis were attenuated by Losartan or by the Caspase 3 inhibitor ZVAD-fmk. Conclusions: These data demonstrate the presence of a functional ANG II and Caspase 3 dependent apoptotic pathways in newborn meconium-instilled lungs. They also imply that meconium-induced apoptosis is modulated by the pulmonary RASL system in which ANG II plays a critical role. Both losartan and the Caspase 3 inhibitor ZVAD-fmk pretreatment significantly decreased meconium-induced Caspase-3 activation and lung cell apoptosis.

N-acetylcysteine protects alveolar epithelial cells from hydrogen peroxide–induced apoptosis through scavenging reactive oxygen species and suppressing c-Jun N-terminal kinase

ABSTRACTThe production of reactive oxygen species (ROS) during hyperoxia contribute to alveolar epithelial apoptosis. In the present study, the molecular mechanisms of oxidative stress–induced alveolar epithelial cell apoptosis were investigated. The cytoprotective effects of N-acetylcysteine (NAC) were evaluated. Treatments using 500 μM H2O2 can induce primary alveolar type II epithelial cell apoptosis. During this procedure, c-Jun N-terminal kinase (JNK) was activated. SP600125, a specific inhibitor of JNK, can partially block H2O2-induced alveolar type II epithelial cells (ATII cells). SP600125 also attenuated Bax protein content and p53 nuclear accumulation induced by H2O2. NAC (5 mM) pretreatment decreased H2O2-induced ATII cell apoptosis. The high level of intracellular reactive oxygen species (ROS) induced by H2O2 was also attenuated by NAC pretreatment. Taken together, H2O2 can induce primary ATII cells apoptosis and increase JNK phosphorylation. NAC, a precursor of glutathione (GSH) synthesis, can protect ATII cells from H2O2-induced apoptosis through scavenging ROS.

Febrile-Range Hyperthermia Augments Lipopolysaccharide-Induced Lung Injury by a Mechanism of Enhanced Alveolar Epithelial Apoptosis

Fever is common in critically ill patients and is associated with worse clinical outcomes, including increased intensive care unit mortality. In animal models, febrile-range hyperthermia (FRH) worsens acute lung injury, but the mechanisms by which this occurs remain uncertain. We hypothesized that FRH augments the response of the alveolar epithelium to TNF-alpha receptor family signaling. We found that FRH augmented LPS-induced lung injury and increased LPS-induced mortality in mice. At 24 h, animals exposed to hyperthermia and LPS had significant increases in alveolar permeability without changes in inflammatory cells in bronchoalveolar lavage fluid or lung tissue as compared with animals exposed to LPS alone. The increase in alveolar permeability was associated with an increase in alveolar epithelial apoptosis and was attenuated by caspase inhibition with zVAD.fmk. At 48 h, the animals exposed to hyperthermia and LPS had an enhanced lung inflammatory response. In murine lung epithelial cell lines (MLE-15, LA-4) and in primary type II alveolar epithelial cells, FRH enhanced apoptosis in response to TNF-alpha but not Fas ligand. The increase in apoptosis was caspase-8 dependent and associated with suppression of NF-kappaB activity. The FRH-associated NF-kappaB suppression was not associated with persistence of IkappaB-alpha, suggesting that FRH-mediated suppression of NF-kappaB occurs by means other than alteration of IkappaB-alpha kinetics. These data show for the first time that FRH promotes lung injury in part by increasing lung epithelial apoptosis. The enhanced apoptotic response might relate to FRH-mediated suppression of NF-kappaB activity in the alveolar epithelium with a resultant increase in susceptibility to TNF-alpha-mediated cell death.