Lipopolysaccharide-induced cytokine expression in alveolar epithelial cells: role of PKCζ-mediated p47phox phosphorylation

Chronic inflammation incited by bacteria in the saccular lung of premature infants contributes to the pathogenesis of bronchopulmonary dysplasia (BPD). LPS-mediated type II alveolar epithelial cell (AEC) injury induces the expression of pro-inflammatory cytokines that trigger pulmonary neutrophil influx, alveolar matrix degradation and lung remodeling. We hypothesized that NADPH oxidase (Nox)-dependent mechanisms mediate LPS-induced cytokine expression in AEC. We examined the role of p47phox in mediating LPS-dependent inflammatory cytokine expression in A549 cells (which exhibit phenotypic features characteristic of type II AEC) and elucidated the proximal signaling events by which Nox is activated by LPS. LPS-induced ICAM-1 and IL-8 expression was associated with increased superoxide formation in AEC. LPS-mediated oxidative stress and cytokine expression was inhibited by apocynin and augmented by PMA demonstrating that Nox-dependent redox signaling regulates LPS-dependent pro-inflammatory signaling in AEC. In LPS-treated cells, p47phox translocated from the cytoplasm to the perinuclear region and co-localized with gp91phox. LPS also induced a temporal increase in p47phox serine304 phosphorylation in AEC. While inhibition of classical PKC and novel PKC with calphostin and rottlerin did not inhibit ICAM-1 or IL-8 expression, the myristolyated PKCζ pseudosubstrate peptide (a specific inhibitor of PKCζ) inhibited LPS-induced cytokine expression in AEC. Inhibition of PKCζ also attenuated LPS-mediated p47phox phosphorylation and perinuclear translocation in AEC. Consistent with these data, LPS activated PKCζ in AEC as evidenced by increased threonine410 phophorylation. We conclude that PKCζ-mediated p47phox activation regulates LPS-dependent cytokine expression in AEC. Selective inhibition of PKCζ or p47phox might attenuate LPS-mediated inflammation and alveolar remodeling in BPD.     

CGRP is essential for protection against alveolar epithelial cell necroptosis by activating the AMPK/L-OPA1 signaling pathway during acute lung injury

Alveolar epithelial cell (AEC) necroptosis is critical to disrupt the alveolar barrier and provoke acute lung injury (ALI). Here, we define calcitonin gene-related peptide (CGRP), the most abundant endogenous neuropeptide in the lung, as a novel modulator of AEC necroptosis in lipopolysaccharide (LPS)-induced ALI. Upon LPS-induced ALI, overexpression of Cgrp significantly mitigates the inflammatory response, alleviates lung tissue damage, and decreases AEC necroptosis. Similarly, CGRP alleviated AEC necroptosis under the LPS challenge in vitro. Previously, we identified that long optic atrophy 1 (L-OPA1) deficiency mediates mitochondrial fragmentation, leading to AEC necroptosis. In this study, we discovered that CGRP positively regulated mitochondrial fusion through stabilizing L-OPA1. Mechanistically, we elucidate that CGRP activates AMP-activated protein kinase (AMPK). Furthermore, the blockade of AMPK compromised the protective effect of CGRP against AEC necroptosis following the LPS challenge. Our study suggests that CRGP-mediated activation of the AMPK/L-OPA1 axis may have potent therapeutic benefits for patients with ALI or other diseases with necroptosis.     

Hyperoxia-induced apoptosis and Fas/FasL expression in lung epithelial cells

Alveolar epithelial apoptosis is an important feature of hyperoxia-induced lung injury in vivo and has been described in the early stages of bronchopulmonary dysplasia (chronic lung disease of preterm newborn). Molecular regulation of hyperoxia-induced alveolar epithelial cell death remains incompletely understood. In view of functional involvement of Fas/FasL system in physiological postcanalicular type II cell apoptosis, we speculated this system may also be a critical regulator of hyperoxia-induced apoptosis. The aim of this study was to investigate the effects of hyperoxia on apoptosis and apoptotic gene expression in alveolar epithelial cells. Apoptosis was studied by TUNEL, electron microscopy, DNA size analysis, and caspase assays. Fas/FasL expression was determined by Western blot analysis and RPA. We determined that in MLE-12 cells exposed to hyperoxia, caspase-mediated apoptosis was the first morphologically and biochemically recognizable mode of cell death, followed by necrosis of residual adherent cells. The apoptotic stage was associated with a threefold upregulation of Fas mRNA and protein expression and increased susceptibility to direct Fas receptor activation, concomitant with a threefold increase of FasL protein levels. Fas gene silencing by siRNAs significantly reduced hyperoxia-induced apoptosis. In murine fetal type II cells, hyperoxia similarly induced markedly increased Fas/FasL protein expression, confirming validity of results obtained in transformed MLE-12 cells. Our findings implicate the Fas/FasL system as an important regulator of hyperoxia-induced type II cell apoptosis. Elucidation of regulation of hyperoxia-induced lung apoptosis may lead to alternative therapeutic strategies for perinatal or adult pulmonary diseases characterized by dysregulated type II cell apoptosis.     

Bleomycin-induced apoptosis of alveolar epithelial cells requires angiotensin synthesis de novo

Primary cultures of rat type II alveolar epithelial cells (AECs) or human AEC-derived A549 cells, when exposed to bleomycin (Bleo), exhibited concentration-dependent apoptosis detected by altered nuclear morphology, fragmentation of DNA, activation of caspase-3, and net cell loss over time. In both cell culture models, exposure to Bleo caused time-dependent increases in angiotensinogen (ANGEN) mRNA. Antisense oligonucleotides against ANGEN mRNA inhibited Bleo-induced apoptosis of rat AEC or A549 cells by 83 and 84%, respectively (P < 0.01 and P < 0.05), and prevented Bleo-induced net cell loss. Apoptosis of rat AECs or A549 cells in response to Bleo was inhibited 91% by the ANG-converting enzyme inhibitor captopril or 82%, respectively, by neutralizing antibodies specific for ANG II (both P < 0.01). Antagonists of ANG receptor AT(1) (losartan, L-158809, or saralasin), but not an AT(2)-selective blocker (PD-123319), inhibited Bleo-induced apoptosis of either rat AECs (79%, P < 0.01) or A549 cells (83%, P < 0.01) and also reduced the activity of caspase-3 by 52% (P < 0.05). These data indicate that Bleo, like Fas(L) or TNF-alpha, induces transactivation of ANG synthesis de novo that is required for AEC apoptosis. They also support the theory that ANG system antagonists have potential for the blockade of AEC apoptosis in situ.     

The microRNA miR-17 regulates lung FoxA1 expression during lipopolysaccharide-induced acute lung injury

Acute lung injury (ALI) is a severe pulmonary disease that causes a high number of fatalities worldwide. Studies have shown that FoxA1 expression is upregulated during ALI and may play an important role in ALI by promoting the apoptosis of alveolar type II epithelial cells. However, the mechanism of FoxA1 overexpression in ALI is unclear. In this study, an in vivo murine model of ALI and alveolar type II epithelial cells injury was induced using lipopolysaccharide (LPS). LPS upregulated FoxA1 in the lung tissue of the in vivo ALI model and in LPS-challenged type II epithelial cells. In contrast, miR-17 was significantly downregulated in these models. After miR-17 antagomir injection, the expression of FoxA1 was significantly increased in ALI mice. MiR-17 mimics could significantly inhibit FoxA1 mRNA and protein expression, whereas the miR-17 inhibitor could significantly increase FoxA1 mRNA and protein expression in LPS-induced type II epithelial cells. Thus, our results suggest that the downregulation of miR-17 expression could lead to FoxA1 overexpression in ALI.     

Protectin DX promotes epithelial injury repair and inhibits fibroproliferation partly via ALX/PI3K signalling pathway

Acute respiratory distress syndrome/acute lung injury (ARDS/ALI) is histologically characterized by extensive alveolar barrier disruption and excessive fibroproliferation responses. Protectin DX (PDX) displays anti‐inflammatory and potent inflammation pro‐resolving actions. We sought to investigate whether PDX attenuates LPS (lipopolysaccharide)‐induced lung injury via modulating epithelial cell injury repair, apoptosis and fibroblasts activation. In vivo, PDX was administered intraperitoneally (IP) with 200 ng/per mouse after intratracheal injection of LPS, which remarkedly stimulated proliferation of type II alveolar epithelial cells (AT II cells), reduced the apoptosis of AT II cells, which attenuated lung injury induced by LPS. Moreover, primary type II alveolar cells were isolated and cultured to assess the effects of PDX on wound repair, apoptosis, proliferation and transdifferentiation in vitro. We also investigated the effects of PDX on primary rat lung fibroblast proliferation and myofibroblast differentiation. Our result suggests PDX promotes primary AT II cells wound closure by inducing the proliferation of AT II cells and reducing the apoptosis of AT II cells induced by LPS, and promotes AT II cells transdifferentiation. Furthermore, PDX inhibits transforming growth factor‐β₁ (TGF‐β₁) induced fibroproliferation, fibroblast collagen production and myofibroblast transformation. Furthermore, the effects of PDX on epithelial wound healing and proliferation, fibroblast proliferation and activation partly via the ALX/ PI3K signalling pathway. These data present identify a new mechanism of PDX which targets the airway epithelial cell and fibroproliferation are potential for treatment of ARDS/ALI.     

Fas/FasL-mediated apoptosis in perinatal murine lungs

Postcanalicular lung development is characterized by a time-specific increase in alveolar epithelial type II cell apoptosis. We have previously demonstrated that, in fetal rabbits, developmental type II cell apoptosis coincides with transient upregulation of the cell death regulator Fas ligand (FasL). The aims of this study were 1) to determine the spatiotemporal patterns of pulmonary apoptosis and Fas/FasL gene expression in the murine model [embryonic day 17 (E17) through postnatal day 5 (P5)], and 2) to investigate the functional involvement of the Fas/FasL system by determining the effect of Fas activation and inhibition on perinatal pulmonary apoptosis. The apoptotic activity of alveolar epithelial type II cells, determined by combined TUNEL labeling and anti-surfactant protein B immunohistochemistry, showed a dramatic increase during the perinatal transition (type II cell apoptotic index <0.1% at E17, 1.5% at P1-P3, and 0.3% at P5). This timing of enhanced type II cell apoptosis coincided with a robust 14-fold increase in Fas mRNA and protein levels and a threefold increase in FasL protein levels; both Fas and FasL immunolocalized to type II and bronchial epithelial cells. In vitro and in vivo exposure of fetal and postnatal murine type II cells to anti-Fas antibody induced a fourfold increase in apoptotic activity that was prevented by administration of a broad-spectrum caspase inhibitor; the pulmonary apoptotic activity of Fas-deficient lpr mice remained unchanged. Conversely, administration of a caspase inhibitor to newborn mice (P1) resulted in marked diminution of pulmonary apoptotic activity. These combined findings strongly implicate the Fas/FasL system as a critical regulator of perinatal type II cell apoptosis. The developmental time dependence of apoptosis-related events in the murine model should facilitate investigations of the regulation of perinatal pulmonary apoptotic gene expression.     

Fas ligand expression coincides with alveolar cell apoptosis in late-gestation fetal lung development

Apoptosis plays a central role in the cellular remodeling of the developing lung. We determined the spatiotemporal patterns of the cell death regulators Fas and Fas ligand (FasL) during rabbit lung development and correlated their expression with pulmonary and type II cell apoptosis. Fetal rabbit lungs (25-31 days gestation) were assayed for apoptotic activity by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) and DNA size analysis. Fas and FasL expression were analyzed by RT-PCR, immunoblot, and immunohistochemistry. Type II cell apoptosis increased significantly on gestational day 28; the type II cell apoptotic index increased from 0.54 +/- 0.34% on gestational day 27 to 3.34 +/- 1.24% on day 28, P < 0.01 (ANOVA). This corresponded with the transition from the canalicular to the terminal sac stage of development. The day 28 rise in epithelial apoptosis was synchronous with a robust if transient 20-fold increase in FasL mRNA and a threefold increase in FasL protein levels. In contrast, Fas mRNA levels remained constant, suggestive of constitutive expression. Fas and FasL proteins were immunolocalized to alveolar type II cells and bronchiolar Clara cells. The correlation of this highly specific pattern of FasL expression with alveolar epithelial apoptosis and remodeling implicates the Fas/FasL system as a potentially important regulatory pathway in the control of postcanalicular alveolar cytodifferentiation.     

Losartan, an antagonist of AT1 receptor for angiotensin II, attenuates lipopolysaccharide-induced acute lung injury in rat

Angiotensin II (Ang II) plays an important role in inflammatory process. Acute lung injury (ALI), an inflammatory disorder of the lung, is commonly associated with endotoxemia; however, the mechanism that endotoxin (lipopolysaccharide, LPS) induces the inflammatory response in ALI is not well defined. Here, we showed, in LPS-induced ALI rat model, that Ang II and Ang II type 1 (AT₁) receptor were significantly increased in lung tissues, compared with those in controls. Meanwhile, nuclear factor (NF)-κB-DNA-binding activity, tumor necrosis factor (TNF)-α mRNA, and pneumocytic apoptosis were significantly increased. Moreover, pretreatment of rats with losartan, an antagonist of AT₁ receptor for Ang II, improved the inflammation, reduced the elevation of Ang II and AT₁ receptor, and inhibited NF-κB-DNA-binding activity, expression of TNF-α mRNA, and pneumocytic apoptosis. The data indicate that Ang II may mediate the inflammatory process in LPS-induced ALI through AT₁ receptor, which can be blocked by losartan.     

Angiotensin II type-1 receptor antagonist attenuates LPS-induced acute lung injury

Angiotensin II is able to trigger inflammatory responses through an angiotensin II type 1 (AT1) receptor. The role of AT1 receptor in acute lung injury (ALI) is poorly understood. Mice were randomly divided into three groups (n = 40 each groups): NS group; LPS group (2 mg/kg LPS intratracheally); and LPS + ZD 7155 group, 10 mg/kg ZD 7155 (an AT1 receptor antagonist) intraperitoneally 30 min prior to LPS exposure. Samples from the lung were isolated and assayed for histopathology analyses or proinflammatory gene expressions, angiotensin II receptors expressions and nuclear factors activities. LPS exposure resulted in severe ALI, elevated levels of TNF-α and IL-1β mRNA expressions, and increased activities of NF-κB and activated protein (AP)-1. Upregulation of AT1 receptor and down-regulation of AT2 receptor were also observed after LPS challenge. Pretreatment with ZD 7155 significantly inhibited the increase of AT1 receptor expression and upregulated AT2 receptor expression. ZD 7155 also reduced the mRNA expression of TNF-α and IL-1β, inhibited the activation of NF-κB and AP-1, and improved lung histopathology. These findings suggest that antagonism of AT1 receptor inhibits the activation of NF-κB and AP-1 in the lung, which may mediate the release of TNF-α and IL-1β and contribute to LPS-induced ALI.     

Protective effects of recombinant 53-kDa protein of Trichinella spiralis on acute lung injury in mice via alleviating lung pyroptosis by promoting M2 macrophage polarization

Trichinella spiralis represents an effective treatment for autoimmune and inflammatory diseases. The effects of recombinant T. spiralis (TS) 53-kDa protein (rTsP53) on acute lung injury (ALI) remain unclear. Here, mice were divided randomly into a control group, LPS group, and rTsP53 + LPS group. ALI was induced in BALB/c mice by LPS (10 mg/kg) injected via the tail vein. rTsP53 (200 µl; 0.4 μg/μl) was injected subcutaneously three times at an interval of 5 d before inducing ALI in the rTsP53+LPS group. Lung pathological score, the ratio and markers of classic activated macrophages (M1) and alternatively activated macrophages (M2), cytokine profiles in alveolar lavage fluid, and pyroptosis protein expression in lung tissue were investigated. RTsP53 decreased lung pathological score. Furthermore, rTsP53 suppressed inflammation by increasing IL-4, IL-10, and IL-13. There was an increase in alveolar M2 macrophage numbers, with an increase in CD206 and arginase-1-positive cells and a decrease in alveolar M1 markers such as CD197 and iNOS. In addition, the polarization of M2 macrophages induced by rTsP53 treatment could alleviate ALI by suppressing lung pyroptosis. RTsP53 was identified as a potential agent for treating LPS-induced ALI via alleviating lung pyroptosis by promoting M2 macrophage polarization.     

Essential role for cathepsin D in bleomycin-induced apoptosis of alveolar epithelial cells

Our earlier studies showed that bleomycin-induced apoptosis of type II alveolar epithelial cells (AECs) requires the autocrine synthesis and proteolytic processing of angiotensinogen into ANG II and that inhibitors of ANG-converting enzyme (ACEis) block bleomycin-induced apoptosis (Li X, Zhang H, Soledad-Conrad V, Zhuang J, and Uhal BD. Am J Physiol Lung Cell Mol Physiol 284: L501-L507, 2003). Given the documented role of cathepsin D (CatD) in apoptosis of other cell types, we hypothesized that CatD might be the AEC enzyme responsible for the conversion of angiotensinogen into ANG I, the substrate for ACE. Primary cultures of rat type II AECs challenged with bleomycin in vitro showed upregulation and secretion of CatD enzymatic activity and immunoreactive protein but no increases in CatD mRNA. The aspartyl protease inhibitor pepstatin A, which completely blocked CatD enzymatic activity, inhibited bleomycin-induced nuclear fragmentation by 76% and reduced bleomycin-induced caspase-3 activation by 47%. Antisense oligonucleotides against CatD mRNA reduced CatD-immunoreactive protein and inhibited bleomycin-induced nuclear fragmentation by 48%. A purified fragment of angiotensinogen (F1-14) containing the CatD and ACE cleavage sites, when applied to unchallenged AEC in vitro, yielded mature ANG II peptide and induced apoptosis. The apoptosis induced by F1-14 was inhibited 96% by pepstatin A and 77% by neutralizing antibodies specific for CatD (both P < 0.001). These data indicate a critical role for CatD in bleomycin-induced apoptosis of cultured AEC and suggest that the role(s) of CatD in AEC apoptosis include the conversion of newly synthesized angiotensinogen to ANG II.     

Alveolar epithelial type II cell apoptosis in vivo during resolution of keratinocyte growth factor-induced hyperplasia in the rat

Keratinocyte growth factor (KGF) induces rapid and transient hyperplasia of alveolar epithelial type II cells. We sought to determine components of the apoptotic process involved in the resolution of this hyperplasia and the fate of the apoptotic cells. Rats received intrabronchial instillation of 5 mg KGF/kg body weight or diluent. Lungs were fixed 1, 2, 3, 5, and 7 days later. Apoptosis was identified by TdT-mediated dUTP nick-end labeling (TUNEL), double-labeling for TUNEL and the type II cell marker MNF116, and electron microscopy. Fas, FasL, Bax, Bcl-2, and pro- and active caspase-3 were studied by immunohistochemistry. Changes were quantified by stereology. Cell type specificity was investigated by immunofluorescence double staining. Type II cells exhibited Fas, FasL, Bcl-2, and procaspase-3 irrespective of treatment and time. Immunoelectron microscopy revealed Fas at the apical type II cell membrane. Bax staining was prominent in controls (45-95% of type II cell surface fraction), markedly decreased during hyperplasia at days 2 (20-40%) and 3 (0-10%), and reappeared at day 7 (25-45%) when apoptosis was prominent. Remnants of apoptotic type II cells were incorporated in membrane-bound vacuoles of type II cell neighbors as well as alveolar macrophages. The results indicate that type II cells can enter the Fas/FasL/caspase-3 pathway regulated by Bax and Bcl-2. High Bcl-2:Bax levels favor type II cell survival and a low rate of apoptosis during hyperplasia. Low Bcl-2:Bax levels favor type II cell apoptosis during resolution. Because of time-dependent changes that occur within a short time, the KGF-treated rat lung provides a useful in vivo model to investigate apoptosis in the context of tissue remodeling and repair.     

Depletion of resident alveolar macrophages does not prevent Fas-mediated lung injury in mice

Activation of the Fas/Fas ligand (FasL) system in the lungs results in a form of injury characterized by alveolar epithelial apoptosis and neutrophilic inflammation. Studies in vitro show that Fas activation induces apoptosis in alveolar epithelial cells and cytokine production in alveolar macrophages. The main goal of this study was to determine the contribution of alveolar macrophages to Fas-induced lung inflammation in mice, by depleting alveolar macrophages using clodronate-containing liposomes. Liposomes containing clodronate or PBS were instilled by intratracheal instillation. After 24 h, the mice received intratracheal instillations of the Fas-activating monoclonal antibody Jo2 or an isotype control antibody and were studied 18 h later. The Jo2 MAb induced increases in bronchoalveolar lavage fluid (BALF) total neutrophils, lung caspase-3 activity, and BALF total protein and worsened histological lung injury in the macrophage-depleted mice. Studies in vitro showed that Fas activation induced the release of the cytokine KC in a mouse lung epithelial cell line, MLE-12. These results suggest that the lung inflammatory response to Fas activation is not primarily dependent on resident alveolar macrophages and may instead depend on cytokine release by alveolar epithelial cells.     

Role of CD69 in acute lung injury

AimsCD69 is an early activation marker in lymphocytes and an important signal transmitter in inflammatory processes. However, its role in acute lung injury (ALI) is still unknown. We used a lipopolysaccharide (LPS)-induced mouse model of ALI to study the role of macrophage-surface CD69 in this condition.Main methodsWe investigated bronchoalveolar lavage fluid (BALF) cell subpopulations, myeloperoxidase levels in lung homogenates, lung pathology, and lung oedema in CD69-deficient (CD69^?/?) mice 24 h after LPS instillation. We also determined cytokine/chemokine expression levels in BALF and macrophage culture supernatant from CD69^?/? and wild type (WT) mice. Also, we investigated CD69, keratinocyte-derived chemokine (KC) and macrophage inflammatory protein (MIP)-2 localization in the lungs after LPS administration. Furthermore, we examined the effect of anti-CD69 antibody on LPS-induced cytokine/chemokine release from cultured macrophages.Key findingsOur study shows that intratracheal instillation of LPS-induced neutrophilic infiltration, histopathological changes, myeloperoxidase positivity, and oedema in the lung to a lower degree in CD69^?/? mice than in WT mice. The immunoreactivities for CD69, KC and MIP2 were induced in the lung of WT mice instilled with LPS and were predominantly localized to the macrophages. Moreover, the cytokine/chemokine expression profile between the two genotypes of cultured macrophages in response to LPS was similar to that observed in the BALF. In addition, anti-CD69 antibody inhibited the LPS-induced cytokine/chemokine expression.SignificanceThese results suggest that CD69 on macrophages plays a crucial role in the progression of LPS-induced ALI and may be a potentially useful target in the therapy for ALI.     

Angiotensin II type-1 receptor antagonist attenuates LPS-induced acute lung injury

Angiotensin II is able to trigger inflammatory responses through an angiotensin II type 1 (AT1) receptor. The role of AT1 receptor in acute lung injury (ALI) is poorly understood. Mice were randomly divided into three groups (n = 40 each groups): NS group; LPS group (2 mg/kg LPS intratracheally); and LPS + ZD 7155 group, 10 mg/kg ZD 7155 (an AT1 receptor antagonist) intraperitoneally 30 min prior to LPS exposure. Samples from the lung were isolated and assayed for histopathology analyses or proinflammatory gene expressions, angiotensin II receptors expressions and nuclear factors activities. LPS exposure resulted in severe ALI, elevated levels of TNF-α and IL-1β mRNA expressions, and increased activities of NF-κB and activated protein (AP)-1. Upregulation of AT1 receptor and down-regulation of AT2 receptor were also observed after LPS challenge. Pretreatment with ZD 7155 significantly inhibited the increase of AT1 receptor expression and upregulated AT2 receptor expression. ZD 7155 also reduced the mRNA expression of TNF-α and IL-1β, inhibited the activation of NF-κB and AP-1, and improved lung histopathology. These findings suggest that antagonism of AT1 receptor inhibits the activation of NF-κB and AP-1 in the lung, which may mediate the release of TNF-α and IL-1β and contribute to LPS-induced ALI.     

miR-146b overexpression ameliorates lipopolysaccharide-induced acute lung injury in vivo and in vitro

Acute respiratory distress syndrome (ARDS) is a type of acute lung injury (ALI), which causes high morbidity and mortality. So far, effective clinical treatment of ARDS is still limited. Recently, miR-146b has been reported to play a key role in inflammation. In the present study, we evaluated the functional role of miR-146b in ARDS using the murine model of lipopolysaccharide (LPS)-induced ALI. The miR-146b expression could be induced by LPS stimulation, and miR-146b overexpression was required in the maintenance of body weight and survival of ALI mice; after miR-146b overexpression, LPS-induced lung injury, pulmonary inflammation, total cell and neutrophil counts, proinflammatory cytokines, and chemokines in bronchial alveolar lavage (BAL) fluid were significantly reduced. The promotive effect of LPS on lung permeability through increasing total protein, albumin and IgM in BAL fluid could be partially reversed by miR-146b overexpression. Moreover, in murine alveolar macrophages, miR-146b overexpression reduced LPS-induced TNF-α and interleukin (IL)-1β releasing. Taken together, we demonstrated that miR-146b overexpression could effectively improve the LPS-induced ALI; miR-146b is a promising target in ARDS treatment.     

Hepatocellular apoptosis is mediated by TNFα-dependent Fas/FasLigand cytotoxicity in a murine model of acute liver failure

There is increasing evidence that the active contribution of hepatocytes to liver disease is strongly dependent on local cytokine environment. It has been shown in vitro that TNFα can enhance hepatocyte FasLigand (FasL)-mediated cytotoxicity. Here, we demonstrate that TNFα-induced apoptosis was associated with Fas and FasL upregulation and that a FasL-neutralizing antibody prevented TNFα-induced apoptosis. We further examined in vivo the relevance of the Fas/FasL pathway to hepatocellular apoptosis in a TNFα-driven model of acute liver failure. Livers of galactosamine/lipopolysaccharide (Gal/LPS)-exposed Fas wild-type mice highly expressed both Fas and FasL and revealed marked hepatocellular apoptosis that was almost completely blocked by soluble TNFα-receptor; this was also almost absent in Gal/LPS-exposed Fas lymphoproliferation mutant mice. Our data provide evidence for a direct link between TNFα and Fas/FasL in mediating hepatocyte apoptosis. Fratricidal death by Fas–FasL interactions of neighbouring hepatocytes may actively contribute to acute liver failure.