LPS-treated Lungs Research Articles

Guettarda crispiflora Vahl Methanol Extract Ameliorates Acute Lung Injury and Gastritis by Suppressing Src Phosphorylation.

Many species in the genus Guettarda are known to exert anti-inflammatory effects and are used as traditional medicinal plants to treat various inflammatory symptoms. However, no studies on the inflammatory activities of Guettarda crispiflora Vahl have been reported. The aim of the study was to investigate in vitro and in vivo the anti-inflammatory effects of a methanol extract of Guettarda crispiflora Vahl (Gc-ME). To determine the anti-inflammatory activity of Gc-ME, lipopolysaccharide (LPS)-, poly(I:C)-, or Pam3CSK4-treated RAW264.7 cells, HCl/EtOH- and LPS-treated mice were employed for in vitro and in vivo tests. LPS-induced nitric oxide production in RAW264.7 cells was determined by Griess assays and cytokine gene expression in LPS-activated RAW264.7 cells, confirmed by RT- and real-time PCR. Transcriptional activation was evaluated by luciferase reporter gene assay. Target protein validation was assessed by Western blot analysis and cellular thermal shift assays (CETSA) with LPS-treated RAW264.7 and gene-transfected HEK293 cells. Using both a HCl/EtOH-induced gastritis model and an LPS-induced lung injury model, inflammatory states were checked by scoring or evaluating gastric lesions, lung edema, and lung histology. Phytochemical fingerprinting of Gc-ME was observed by using liquid chromatography-mass spectrometry. Nitric oxide production induced by LPS and Pam3CSK4 in RAW264.7 cells was revealed to be reduced by Gc-ME. The LPS-induced upregulation of iNOS, COX-2, IL-6, and IL-1β was also suppressed by Gc-ME treatment. Gc-ME downregulated the promotor activities of AP-1 and NF-κB triggered by MyD88- and TRIF induction. Upstream signaling proteins for NF-κB activation, namely, p-p50, p-p65, p-IκBα, and p-Src were all downregulated by Ch-EE. Moreover, Src was revealed to be directly targeted by Gc-ME. This extract, orally treated strongly, attenuated the inflammatory symptoms in HCl/EtOH-treated stomachs and LPS-treated lungs. Therefore, these results strongly imply that Guettarda crispiflora can be developed as a promising anti-inflammatory remedy with Src-suppressive properties.

Cyclooxygenase-2 deficiency attenuates lipopolysaccharide-induced inflammation, apoptosis, and acute lung injury in adult mice.

Many lung diseases are caused by an excessive inflammatory response, and inflammatory lung diseases are often modeled using lipopolysaccharide (LPS) in mice. Cyclooxygenase-2 (COX-2) encoded by the Ptgs2 gene is induced in response to inflammatory stimuli including LPS. The objective of this study was to test the hypothesis that mice deficient in COX-2 (Ptgs2-/-) will be protected from LPS-induced lung injury. Wild-type (WT; CD1 mice) and Ptgs2-/- mice (on a CD1 background) were treated with LPS or vehicle for 24 h. LPS treatment resulted in histological evidence of lung injury, which was attenuated in the Ptgs2-/- mice. LPS treatment increased the mRNA levels for tumor necrosis factor-α, interleukin-10, and monocyte chemoattractant protein-1 in the lungs of WT mice, and the LPS-induced increases in these levels were attenuated in the Ptgs2-/- mice. The protein levels of active caspase-3 and caspase-9 were lower in the LPS-treated lungs of Ptgs2-/- mice than in LPS-treated WT mice, as were the number of terminal deoxynucleotide transferase dUTP nick end labeling-positive cells in lung sections. LPS exposure resulted in a greater lung wet-to-dry weight ratio (W/D) in WT mice, suggestive of pulmonary edema, while in LPS-treated Ptgs2-/- mice, the W/D was not different from controls and less than in LPS-treated WT mice. These results demonstrate that COX-2 is involved in the inflammatory response to LPS and suggest that COX-2 not only acts as a downstream participant in the inflammatory response, but also acts as a regulator of the inflammatory response likely through a feed-forward mechanism following LPS stimulation.

MicroRNA-762 Modulates Lipopolysaccharide-induced Acute Lung Injury via SIRT7

ABSTRACT Background: Inflammation and oxidative stress contribute to the pathogenesis of lipopolysaccharide (LPS)-induced acute lung injury (ALI). MicroRNA-762 (miR-762) has been implicated in the progression of inflammation and oxidative stress; however, its role in ALI remains unclear. In this study, we aim to investigate the role and underlying mechanisms of miR-762 in LPS-induced ALI. Methods: Mice were intravenously injected with miR-762 antagomir, agomir or the negative controls for 3 consecutive days and then received a single intratracheal instillation of LPS (5 mg/kg) for 12 h to establish ALI model. Adenoviral vectors were used to knock down the endogenous SIRT7 expression. Results: An increased miR-762 expression was detected in LPS-treated lungs. miR-762 antagomir significantly reduced inflammation, oxidative stress and ALI in mice, while the mice with miR-762 agomir treatment exhibited a deleterious phenotype. Besides, we found that SIRT7 upregulation was essential for the pulmonoprotective effects of miR-762 antagomir, and that SIRT7 silence completely abolished the anti-inflammatory and anti-oxidant capacities of miR-762 antagomir. Conclusion: miR-762 is implicated in the pathogenesis of LPS-induced ALI via modulating inflammation and oxidative stress, which depends on its regulation of SIRT7 expression. It might be a valuable therapeutic target for the treatment of ALI.

Magnetic Resonance Imaging of Protease-Mediated Lung Tissue Inflammation and Injury.

Pulmonary inflammation usually involves strong neutrophil recruitment with a marked release of proteases such as neutrophil elastase (NE). Noninvasive in vivo assessment of unregulated elastase activity in the lungs would provide a valuable diagnostic tool. Here, it is proposed to use Overhauser-enhanced magnetic resonance imaging (OMRI) in mice where inflammation was induced by the instillation of lipopolysaccharide (LPS). OMRI contrast in the lungs was generated by a dedicated NE free radical substrate. The free radical decayed more rapidly in LPS-treated mouse lungs than in control mice, indicating the occurrence of increased proteolysis under inflammation. Preclinical detection of abnormal proteolysis opens the way for new diagnosis modality and antiprotease testing in vivo.

Loss of Nucleobindin-2/Nesfatin-1 increases lipopolysaccharide-induced murine acute lung inflammation.

NUCB2/nesfatin-1 is expressed in variety of tissues. Treatment with nesfatin-1 reduces inflammation in rat models of subarachnoid hemorrhage-induced oxidative brain damage and traumatic brain injury as well as myocardial injury. There is only one study showing anti-inflammatory actions of nesfatin-1 on acute lung inflammation. To more precisely determine the role of NUCB2/nesfatin-1 in acute lung inflammation, we conducted a study using NUCB2/nesfatin-1 knockout (NKO) mice as well as neutrophils isolated from the bone marrows of WT and NKO mice. Our findings suggest that the absence of NUCB2/nesfatin-1 significantly increases the accumulation of adherent neutrophils by approximately 3 times compared with WT within LPS-treated lungs. Integrating this with observations from both BALF and neutrophil cytokine expression, we propose that although neutrophils lacking NUCB2/nesfatin-1 individually secrete less pro-inflammatory cytokines compared with stimulated WT cells, the result of knocking out NUCB2/nesfatin-1 is net pro-inflammatory. No change was found in NUCB2/nesfatin-1 mRNA or protein expression comparing WT LPS and PBS-treated samples. Taken together, our results show that NUCB2/nesfatin-1 is constitutively expressed in mouse lungs and neutrophils and demonstrates anti-inflammatory properties in mouse lungs during acute lung injury, by inhibiting adherent neutrophil accumulation and inflammatory cytokine expression.

Sectm1a Facilitates Protection against Inflammation-Induced Organ Damage through Promoting TRM Self-Renewal

Tissue-resident macrophages (TRMs) are sentinel cells for maintaining tissue homeostasis and organ function. In this study, we discovered that lipopolysaccharide (LPS) administration dramatically reduced TRM populations and suppressed their self-renewal capacities in multiple organs. Using loss- and gain-of-function approaches, we define Sectm1a as a novel regulator of TRM self-renewal. Specifically, at the earlier stage of endotoxemia, Sectm1a deficiency exaggerated acute inflammation-induced reduction of TRM numbers in multiple organs by suppressing their proliferation, which was associated with more infiltrations of inflammatory monocytes/neutrophils and more serious organ damage. By contrast, administration of recombinant Sectm1a enhanced TRM populations and improved animal survival upon endotoxin challenge. Mechanistically, we identified that Sectm1a-induced upregulation in the self-renewal capacity of TRM is dependent on GITR-activated T helper cell expansion and cytokine production. Meanwhile, we found that TRMs may play an important role in protecting local vascular integrity during endotoxemia. Our study demonstrates that Sectm1a contributes to stabling TRM populations through maintaining their self-renewal capacities, which benefits the host immune response to acute inflammation. Therefore, Sectm1a may serve as a new therapeutic agent for the treatment of inflammatory diseases.

Cytosolic DNA-STING-NLRP3 axis is involved in murine acute lung injury induced by lipopolysaccharide.

The role of NOD‐like receptor protein 3 (NLRP3)‐mediated pyroptosis in acute lung injury (ALI) has been well identified previously. Stimulator of interferon genes (STING) is an indispensable adaptor protein, which could regulate inflammation and pyroptosis during infection; however, its role in lipopolysaccharide (LPS)‐induced ALI remains obscure. This study aimed to explore whether STING participated in the development of LPS‐induced ALI as well as the underlying mechanism. We confirmed that LPS significantly enhanced the expression and phosphorylation of STING in lung tissue and primary macrophages from mice. STING deficiency relieved inflammation and oxidative stress in LPS‐treated murine lungs and macrophages. Meanwhile, STING deficiency also abolished the activation of NLRP3 inflammasome and pyroptosis; however, NLRP3 overexpression by adenovirus offset the beneficial effects of STING deficiency in macrophages treated with LPS. Additionally, the level of mitochondrial DNA (mt‐DNA) significantly increased in macrophages after LPS treatment. Intriguingly, although exogenous mt‐DNA stimulation did not influence the level of STING, it could still trigger the phosphorylation of STING as well as pyroptosis, inflammation, and oxidative stress of macrophages. And the adverse effects induced by mt‐DNA could be offset after STING was knocked out. Furthermore, the inhibition of the sensory receptor of cytosolic DNA (cyclic GMP‐AMP synthase, cGAS) also blocked the activation of STING and NLRP3 inflammasome, meanwhile, it alleviated ALI without affecting the expression of STING after LPS challenge. Furthermore, cGAS inhibition also blocked the production of cGAMP induced by LPS, indicating that mt‐DNA and cGAS could activate STING‐NLRP3‐mediated pyroptosis independent of the expression of STING. Finally, we found that LPS upregulated the expression of transcription factor c‐Myc, which subsequently enhanced the activity of STING promoter and promoted its expression without affecting its phosphorylation. Collectively, our study disclosed that LPS could activate STING in a cytosolic DNA‐dependent manner and upregulate the expression of STING in a c‐Myc‐dependent manner, which cooperatively contribute to ALI.

14 - An inhibitor of NOX2 activation prevents lung injury following LPS administration

We have identified a peptide (called PIP-2) that specifically inhibits the activation of NADPH oxidase, type 2 (NOX2). Mice (C57Bl/6) were administered intratracheal lipopolysaccharide (LPS derived from E.coli) at 5 μg/g body wt. Lungs from mice that were evaluated at 12, 16, or 24 hours after LPS showed inflammation (increased cells and protein in the lung lavage fluid), oxidative stress (increased TBARS, 8-isoprostanes, and protein carbonyls in the lung homogenate ), and lung edema (increased lung wet to dry weight ratio). These indices of acute lung injury (ALI) were stable through 24 hr with partial recovery at 48 hr after LPS. Treatment with PIP-2 at 0, 12, or 16 hr after LPS resulted in a complete resolution of the indices of lung injury when lungs were examined at 24 hr. We interpret these results as a reflection of ongoing injury and repair processes in the lung following LPS. In control LPS-treated lungs, ongoing injury and repair were essentially in balance so that the manifestations of acute lung injury remained constant between 12 and 24 hr after LPS administration. However, with administration of PIP-2 at 12 or 16 hr, inhibition of NOX2 activation allowed the lung to fully repair prior to its examination at 24 hr. Thus, inhibition of NOX2 activation can protect lungs against inflammatory lung injury associated with administration of LPS. The non-toxic PIP-2 peptide could be a useful therapeutic agent to prevent oxidative lung injury.

Circulating soluble endoglin modifies the inflammatory response in mice.

Inflammation is associated with every health condition, and is an important component of many pathologies such as cardiovascular diseases. Circulating levels of soluble endoglin have been shown to be higher in the serum of patients with cardiovascular diseases with a significant inflammatory component. The aim of this study was to evaluate the implication of circulating soluble endoglin in the inflammatory response. For this purpose, a transgenic mouse expressing human soluble endoglin (sEng+) was employed, and three different inflammatory approaches were used to mimic inflammatory conditions in different tissues. This study shows that control sEng+ mice have a normal inflammatory state. The lung and kidney injury induced by the inflammatory agents was reduced in sEng+ mice, especially the intra-alveolar and kidney infiltrates, suggesting a possible reduction in inflammation induced by soluble endoglin. To deepen into this possible effect, the leukocyte number in the bronchoalveolar lavage and air pouch lavage was evaluated and a significant reduction of neutrophil infiltration in LPS-treated lungs and ischemic kidneys from sEng+ with respect to WT mice was observed. Additionally, the mechanisms through which soluble endoglin prevents inflammation were studied. We found that in sEng+ animals the increment of proinflammatory cytokines, TNFα, IL1β and IL6, induced by the inflammatory stimulus was reduced. Soluble endoglin also prevents the augmented adhesion molecules, ICAM, VCAM and E-selectin induced by the inflammatory stimulus. In addition, vascular permeability increased by inflammatory agents was also reduced by soluble endoglin. These results suggest that soluble endoglin modulates inflammatory-related diseases and open new perspectives leading to the development of novel and targeted approaches for the prevention and treatment of cardiovascular diseases.

Therapeutic Potential of Beta-Nicotinamide Adenine Dinucleotide to Reverse Endothelial Barrier Dysfunction in Sickle Cell Mouse Model

Background: Thepulmonary endothelium is a metabolically active monolayer on the luminal surface of the lung vasculature. It regulates a variety of functions including the passage of macromolecules and fluid between the blood and lung interstitial tissue. Inflammatory agents such as bacterial endotoxin lipopolysaccharide (LPS) cause lung endothelial cells (EC) dysfunction leading to pulmonary edema predisposing to acute chest syndrome, a leading cause of death in sickle cell disease (SCD); therefore, a drug which repairs the damaged EC barrier function might be of therapeutic potential. Recently we demonstrated that β-nicotinamide adenine dinucleotide (β-NAD) reverses LPS-induced EC barrier dysfunction in a model of acute lung injury (Exp Lung Res 2012, 38, 223-32). In this study, we investigated the protective effect of β-NAD against the damaging effects of LPS using primary ECs from human and sickle cell mouse lungs as well as survival studies using the preclinical sickle cell mouse model.Methods: Early passaged human lung microvascular EC (HLMVEC) were obtained from Lonza and mouse lung microvascular EC (MLMVEC) were isolated from the Townes knock-in homozygous sickle cell (SS) and heterozygous sickle trait (AS) transgenic mice. The EC barrier function was measured using electrical cell-substrate impedance sensing (ECIS) technique. Western blot was done to measure phosphorylation of proteins involved in the regulation of EC barrier function. Survival studies were performed in SS mice treated with intraperitoneal (IP) LPS (0.05 mg/kg) alone or combined with β-NAD (11 mg/kg); normal saline served as a control. Lungs were harvested at 10-12 hrs from the LPS group and at 24 hrs from saline or LPS/β-NAD groups and stained with Hematoxylin and Eosin for to assess interstitial edema and myeloperoxidase as a marker of neutrophil infiltration.Results: Barrier function measurements using ECIS of HLMVEC indicate that β-NAD (100 µM) enhanced basal EC barrier function by 50-60%. In addition, β-NAD attenuated LPS-induced EC gap formation and actin stress fibers. In human lung endothelium, myosin light chain phosphatase (MLCP) plays a crucial role in the maintenance of the EC barrier integrity. Furthermore, LPS mediates MLCP inactivation by phosphorylating the MLCP regulatory subunit MYPT1 and cytoskeletal targets ERM (Ezrin, Radixin, and Moesin) and CaD (Caldesmon). Western blot analysis demonstrated that β-NAD reversed LPS-induced phosphorylation of MYPT1, ERM and CaD proteins. To determine a functional role, we performed studies using 10 nM MYPT1 siRNA treatment followed by β-NAD (100 μM) for 5 hrs. We observed a 50% attenuation of β-NAD enhanced barrier function suggesting MYPT1 is involved in the mechanism of action for β-NAD. Subsequent studies with SS-MLMVEC demonstrated β-NAD mediated a 40% improvement in barrier function which was comparable to HLMVEC. Further, LPS treatment for 16 hrs produced a 25% decrease in barrier function in SS-MLMVEC compared to a 7% decrease in AS-MLMVEC suggesting impaired EC barrier function in SS lungs. Finally we performed survival studies to determine the ability of β-NAD to protect against LPS-induced mortality in SS mice. In dose optimization studies of LPS (0.1-1.0 mg/kg/dose), SS mice survived for 10-12 hrs with the 0.05 mg/kg LPS dose. By contrary, all LPS doses tested had no effect on AS mice survival. Based on this result, SS mice were given IP injections of saline (3 mice), LPS (4 mice) or LPS/β-NAD (4 mice) and survival rates were monitored. The saline or LPS/β-NAD treated mice survived for 24 hrs (100%) compared with LPS mice which died by 10-12 hrs (p=0.01) supporting a protective effect of β-NAD. Histological assessment of the lung tissue showed increased interstitial edema by Hematoxylin and Eosin stain and neutrophil infiltration by myeloperoxidase in the LPS-treated lungs; these changes were much less prominent in the lungs from LPS/β-NAD treated SS mice.Conclusions: Our data support a role of MLCP and its cytoskeletal targets in the protective effects of β-NAD against LPS-induced HLMVEC barrier dysfunction. Studies using SS-MLMVEC suggest that SS mice have impaired barrier function and greater susceptibility to LPS toxicity. However, β-NAD treatment improved survival of SS mice supporting the therapeutic potential of this agent to treat the complication of acute chest syndrome in SCD. DisclosuresMakala:Georgia Regents University: Employment.

Prostaglandin D2 ameliorates acute lung inflammation by activating Nrf2 independent of 15-deoxy-Δ12,14-PGJ2. (P5040)

Abstract Since anti-inflammatory activity of prostaglandin D2 (PGD2) is attributed to 15-deoxy-Δ12,14-PGJ2, a byproduct of PGD2, we explored the hypothesis that PGD2, independent of 15-deoxy-Δ12,14-PGJ2, activates NF-E2-related factor 2 (Nrf2), an anti-inflammatory transcription factor, contributing to suppression of lung inflammation in acute lung injury (ALI). Intratracheal delivery of PGD2 decreased neutrophilic infiltration, MPO activity, and the production of pro-inflammatory cytokines in LPS-treated lungs. In contrast, lipocalin-prostaglandin D synthase (L-PGDS) KO mice that produce less PGD2 showed prolonged lung inflammation, highlighting the importance of L-PGDS and PGD2 in suppressing lung inflammation. Two log10 less of PGD2 than 15-deoxy-Δ12,14-PGJ2 was sufficient for activating Nrf2 in RAW 264.7. Similarly, PGD2 was more potent than 15-deoxy-Δ12,14-PGJ2 in expressing Nrf2 dependent genes in mouse lungs. While overexpression of DP1, a receptor of PGD2, enhanced PGD2-mediated Nrf2 activation, siRNA silencing DP1 expression reduced its activation, suggesting that PGD2 uses DP1 in activating Nrf2. Furthermore, PGD2 synergistically suppressed neutrophil infiltration when nrf2 was functionally complemented to the lung of the nrf2-/- mice. Our results show that PGD2 activated Nrf2 independent of 15-deoxy-Δ12,14-PGJ2 and involved DP1. We suggest that PGD2 has a potent anti-inflammatory activity, which is at least in part mediated by activating Nrf2.

Lipopolysaccharide-induced injury is more pronounced in fetal transgenic ErbB4-deleted lungs

Pulmonary ErbB4 deletion leads to a delay in fetal lung development, alveolar simplification, and lung function disturbances in adult mice. We generated a model of intrauterine infection in ErbB4 transgenic mice to study the additive effects of antenatal LPS administration and ErbB4 deletion during fetal lung development. Pregnant mice were treated intra-amniotically with an LPS dose of 4 μg at E17 of gestation. Lungs were analyzed 24 h later. A significant influx of inflammatory cells was seen in all LPS-treated lungs. In heterozygote control lungs, LPS treatment resulted in a delay of lung morphogenesis characterized by a significant increase in the fraction of mesenchyme, a decrease in gas exchange area, and disorganization of elastic fibers. Surfactant protein (Sftp)b and Sftpc were upregulated, but mRNA of Sftpb and Sftpc was downregulated compared with non-LPS-treated controls. The mRNA of Sftpa1 and Sftpd was upregulated. In ErbB4-deleted lungs, the LPS effects were more pronounced, resulting in a further delay in morphological development, a more pronounced inflammation in the parenchyma, and a significant higher increase in all Sftp. The effect on Sftpb and Sftpc mRNA was somewhat different, resulting in a significant increase. These results imply a major role of ErbB4 in LPS-induced signaling in structural and functional lung development.

Mitochondrial donation by mesenchymal stromal cells rescues alveolar surfactant secretion in sepsis

In sepsis, bone marrow derived mesenchymal stromal cells (MSCs) protect against acute lung injury (ALI). However, underlying mechanisms are unclear. We reported, MSCs transfer mitochondria to cultured capillaries (Otsu et al, 2008). To determine whether mitochondrial transfer occurs in situ, in anesthetized rats we intratracheally instilled lipopolysaccharide (LPS) or saline. After 4 hours, we instilled MSCs (2×106 cells) that expressed the mitochondria‐targeted fluorescent protein, DsRed. After 24 hours, we isolated and blood‐perfused the lungs. In alveoli viewed by confocal microscopy, we determined DsRed fluorescence and alveolar surfactant secretion in single type 2 (AT2) cells (Ashino et al, 2000). In saline‐treated lungs, no alveolar MSCs were evident. In LPS‐treated lungs, in some but not all alveoli MSCs adhered to alveolar epithelium (AE) and DsRed localized intracellularly in AT2 cells. Surfactant secretion stimulated by a 15‐second hyperinflation, was evident in alveoli containing MSCs, but not in MSC‐free alveoli. Our findings indicate that in LPS‐treated lungs (i) MSCs transfer mitochondria to AT2 cells, and that (ii) surfactant secretion is abrogated in MSC‐free alveoli, but protected in alveoli containing MSCs. MSC‐AEC mitochondrial transfer might increase ATP in AT2 cells, thereby rescuing surfactant secretion and protecting lung function in ALI (support HL64896 & HL57556).

TLR4/MyD88-induced non-migratory myeloid cells suppress Th2 effector function in the lung (91.15)

Abstract Bacterial lipopolysaccharide (LPS) is immunostimulatory at a low dose but immunosuppressive at a high dose in the respiratory tract as observed in animal studies. Relevant to LPS-mediated immunosuppression in the lungs of animals is the protective effect of high doses of LPS in allergic diseases such as asthma in humans. Our study investigated the impact of a high dose of LPS on cells of the innate immune system in the lung to elucidate the basis of LPS-mediated immunosuppression. A high LPS dose blunted house dust mite-induced airway eosinophilia and Th2 cytokines production. Conversely, adoptively transferred Th2 cells were unable to mount allergic airway inflammation in LPS-treated lungs. The high LPS dose promoted the development of a lung-resident myeloid cell type in a TLR4- and MyD88-dependent fashion that suppressed lung DC-mediated reactivation of primed Th2 cells. Adoptive transfer of these myeloid cells suppressed allergen-induced airway inflammation. These data suggest that LPS-induced non-migratory myeloid cells might contribute to the protective effects of LPS in allergic airways disease by dampening Th2 effector functions in the lung. RO1 HL060207 (P.R.), RO1 HL077430 (A.R.), RO1 HL084932 (A.R. and P.R.) and AHA 0865379D (M.A.)

EXPRESSION AND ACTIVITIES OF N-MYRISTOYLTRANSFERASE AND CALCINEURIN IN NORMAL AND INFLAMED LUNGS

The role of N-myristoyltransferase and calcineurin is well established in signaling pathways. However, there are no data on their expression and activities in normal and inflamed lungs. The mechanisms of lung inflammation induced following administration of lipopolysaccharides (LPS) or exposure to swine barn air remain unclear. Therefore, we examined expression and activities of N-myristoyltransferase and calcineurin in normal and inflamed lungs of rats. Histopathology showed acute inflammation in the lungs of rats exposed to barn air or LPS but not of control rats. There was no difference in the activities of N-myristoyltransferase and calcineurin among the control, barn-exposed, and LPS-treated rat lungs. Although N-myristoyltransferase and calcineurin were localized in airway epithelium, blood vessel walls, alveolar macrophages, and septa in the lungs of rats from all the groups, the staining intensity was increased in the lungs from rats exposed to intravenous LPS or barn air. Densitometric analyses of Western blots of 55- and 60-kDa polypeptide bands corresponding to N-myristoyltransferase and calcineurin, respectively, in the lung homogenates revealed no differences among the groups. These results show that expression of myristoyltransferase and calcineurin in lung epithelium and endothelium and a cell-specific increase in immunohistochemical expression.

Impaired mitochondrial Ca2+ dynamics in lipopolysaccharide‐treated lungs

Mitochondrial Ca2+ determines exocytosis in lung (J. Clin. Invest. 2003; 111:–699). To define surfactant secretion mechanisms in a sepsis model, in anesthetized rats we intra-tracheally instilled saline (control; n=4), or lipopolysaccharide (LPS, 1 mg/kg; n=4). After 24 h, we isolated and blood-perfused lungs at constant pulmonary artery, left atrial and alveolar (Palv) pressures of 10, 3 and 5 cmH2O, respectively. We viewed single alveoli by epi-fluorescence microscopy. By alveolar micropuncture we loaded the mitochondrial Ca2+ dye, rhod 2, or the lamellar body (LB)-staining dye, LysoTracker Red (LTR). Type 2 cells evoked strong LTR fluorescence (grey levels). In control lungs, a single 15-s inflation to Palv of 15 cmH2O increased rhod 2 fluorescence by 42.4±10.2% (mean±SE; P<0.05), indicating increase of mitochondrial Ca2+. Concomitantly, LTR fluorescence decreased 77.5±9.1% (P<0.05), indicating onset of LB exocytosis. By contrast, in LPS-treated lungs, a similar inflation decreased rhod 2 fluorescence by 20.4±4.8% (P<0.05), but LTR fluorescence failed to change from baseline. Hence, in LPS-treated lungs, inflation decreased mitochondrial Ca2+ and failed to induce surfactant secretion. These data are the first direct evidence that lung exposure to LPS causes surfactant failure, possibly secondary to dysfunctional mitochondrial Ca2+ regulation (Support: HL64896).

Transcriptional Profiling of Lipopolysaccharide-Induced Acute Lung Injury

Mortality associated with acute lung injury (ALI) induced by lipopolysaccharide (LPS) remains high in humans, warranting improved treatment and prevention strategies. ALI is characterized by the expression of proinflammatory mediators and extensive neutrophil influx into the lung, followed by severe lung damage. Understanding the pathogenesis of LPS-induced ALI is a prerequisite for designing better therapeutic strategies. In the present study, we used microarrays to gain a global view of the transcriptional responses of the lung to LPS in a mouse model of ALI that mimics ALI in humans. A total of 71 inflammation-associated genes were up-regulated in LPS-treated lungs, including a chemokine, LPS-induced CXC chemokine (LIX), whose role in the induction of ALI is unknown. Most of the inflammatory genes peaked at 2 h post-LPS treatment. Real-time reverse transcription-PCR confirmed the LPS-induced up-regulation of selected genes identified by microarray analysis, including LIX. The up-regulation of LIX, tumor necrosis factor alpha, and macrophage inflammatory protein 2 was confirmed at the protein level by enzyme-linked immunosorbent assays. To determine the role of LIX in the induction of ALI, we used both exogenous LIX and a LIX blocking antibody. Exogenous LIX alone elicited a neutrophil influx in the lungs, and the anti-LIX antibody attenuated the LPS-induced neutrophil accumulation in the lungs. Taken together, the results of our study demonstrate for the first time the temporal expression of inflammatory genes during LPS-induced ALI and suggest that early therapeutic intervention is crucial to attenuate lung damage. Moreover, we identified a role for LIX in the induction of ALI, and therefore LIX may serve as a novel therapeutic target for the minimization of ALI.

L-Lysine Decreases Nitric Oxide Production and Increases Vascular Resistance in Lungs Isolated from Lipopolysaccharide-Treated Neonatal Pigs

Nitric oxide (NO) production may depend on the uptake of L-arginine (L-arg), the substrate for NO synthase in inflammatory lung diseases. The cellular transport of L-arg occurs via the cationic amino acid transporters (CAT), and L-lysine (L-lys) competitively inhibits CAT. Neonatal pigs were treated with lipopolysaccharide (LPS) or vehicle for 4 h. LPS increased exhaled NO (exNO; 0.026 +/- 0.003 to 0.046 +/- 0.003 nmol. kg(-1). min(-1); p < 0.005) and decreased mean systemic arterial blood pressure (89 +/- 4 to 67 +/- 4 mm Hg; p < 0.05), whereas vehicle did not affect exNO or mean systemic arterial blood pressure. The lungs were then isolated and perfused; exNO was greater in lungs from LPS-treated animals (0.08 +/- 0.01 nmol/kg/min) than in lungs from vehicle-treated animals (0.05 +/- 0.01 nmol. kg(-1). min(-1); p < 0.05). The addition of L-arg (0.3 mM) significantly (p < 0.05) increased exNO production in both groups of lungs (mean increase 0.04 +/- 0.01 nmol. kg(-1). min(-1) LPS-treated lungs, p < 0.05; mean increase 0.02 +/- 0.01 nmol. kg(-1). min(-1) vehicle-treated lungs); however, L-arg decreased pulmonary vascular resistance (PVR) only in LPS-treated lungs (mean decrease 0.03 +/- 0.01 mm Hg. ml(-1). kg(-1). min(-1), p < 0.05). L-lys caused a dose-dependent decrease in exNO production and a dose-dependent increase in PVR in LPS-treated lungs. L-lys decreased exNO only at 30 mM and had no effect on PVR in vehicle-treated lungs. In four lungs each from vehicle- and LPS-treated animals, reverse transcriptase-PCR demonstrated CAT-2 mRNA only in LPS-treated animals. These results suggest that the increased NO production in the lungs from LPS-treated animals depends on the uptake of vascular L-arg.

Surfactant protein A differentially regulates peripheral and inflammatory neutrophil chemotaxis.

Surfactant protein A (SP-A), a pulmonary lectin, plays an important role in regulating innate immune cell function. Besides accelerating pathogen clearance by pulmonary phagocytes, SP-A also stimulates alveolar macrophage chemotaxis and directed actin polymerization. We hypothesized that SP-A would also stimulate neutrophil chemotaxis. With the use of a Boyden chamber assay, we found that SP-A (0.5-25 microg/ml) did not stimulate chemotaxis of rat peripheral neutrophils or inflammatory bronchoalveolar lavage (BAL) neutrophils isolated from LPS-treated lungs. However, SP-A affected neutrophil chemotaxis toward the bacterial peptide formyl-met-leu-phe (fMLP). Surprisingly, the effect was different for the two neutrophil populations: SP-A reduced peripheral neutrophil chemotaxis toward fMLP (49 +/- 5% fMLP alone) and enhanced inflammatory BAL neutrophil chemotaxis (277 +/- 48% fMLP alone). This differential effect was not seen for the homologous proteins mannose binding lectin and complement protein 1q but was recapitulated by type IV collagen. SP-A bound both neutrophil populations comparably and did not alter formyl peptide binding. These data support a role for SP-A in regulating neutrophil migration in pulmonary tissue.

Clearance of surfactant lipids by neutrophils and macrophages isolated from the acutely inflamed lung.

Pulmonary surfactant reduces surface tension at the lung air-liquid interface and defends the host against infection. Several lines of evidence show that surfactant levels are altered in animal models and patients with inflammatory or infectious lung diseases. We tested the hypothesis that cells responding to lung injury alter surfactant levels through increased phospholipid clearance. Acute lung injury was induced by intratracheal administration of lipopolysaccharide (LPS; Escherichia coli 026:B6) into rats. LPS exposure resulted in a 12-fold increase in the number of cells isolated by lavage, the majority of which were neutrophils. Isolated macrophages and neutrophils from LPS-treated lungs internalized and degraded lipids in vitro, and LPS injury stimulated uptake by macrophages twofold. We estimate that lipid clearance by lavage cells in LPS-treated lungs could be enhanced 6- to 13-fold with both activated macrophages and increased numbers of neutrophils contributing to the process. These data show that the increased number of cells in the alveolar space after acute lung injury may lead to alterations in surfactant pools via enhanced clearance and degradation of lipids.