Stretch and CO2 modulate the inflammatory response of alveolar macrophages through independent changes in metabolic activity

Ventilatory-induced strain can exacerbate acute lung injury (ALI). Current ventilation strategies favour low tidal volumes and high end-expiratory volumes to 'rest' the lung, but can lead to an increase in CO2. Alveolar macrophages (AM) play a pivotal role in ALI through the release of inflammatory mediators. The effect of physical strain and CO2 on the release of pro-inflammatory mediators was examined in isolated rat AM. AM were cultured on IgG-coated silastic membranes with or without lipopolysaccharide (LPS) and 5% or 20% CO2 and subjected to a repetitive sinusoidal mechanical strain (30%, 60 cycles/min) for 4 h. Cell viability and metabolic activity were assessed. In both the presence and absence of LPS, physical strain increased metabolic activity by approximately 5%, while 20% CO2 decreased metabolic activity by approximately 40%. Twenty per cent CO2 decreased TNF-alpha secretion by approximately 45%, without affecting cell viability. Physical strain enhanced LPS-induced secretion of TNF-alpha by 1.5%, but not IL-6 or CINC-1. Hence, the effects of both CO2 and physical strain are mediated independently through changes in AM metabolic activity. Physical strain is not a major determinant of TNF-alpha, IL-6 or CINC-1 in AM. Our results confirm that high CO2 can lessen the TNF-alpha inflammatory response of AM.     

Differential regulation of tumor necrosing factor-alpha (TNF-alpha) and interleukin-10 (IL-10) secretion by protein kinase and phosphatase inhibitors in human alveolar macrophages.

IL-10, a cytokine first identified as a product of cloned Th2 lymphocytes, is also produced by monocytes/macrophages. By its ability to inhibit cytokine synthesis and the expression of surface antigens, IL-10 is able to temper inflammation. In contrast, TNF-alpha plays a key role in acute and chronic inflammation and has been implicated in several forms of lung injury. The objective of this study was to investigate whether activators or inhibitors of LPS-activated signalling pathways might be able to dissociate TNF-alpha from IL-10 secretion in alveolar macrophages (AM). The results show that PMA activates expression of TNF-alpha without inducing IL-10 expression. We further demonstrate that LPS-induced TNF-alpha secretion is independent of PKC activation and can be increased by inhibitors of the serine/threonine phosphatases PP1 and PP2A. In contrast, LPS-mediated IL-10 secretion is down-regulated by PMA and inhibitors of PP1 and PP2A. Addition of PKC inhibitors reverses the PMA-mediated down-regulation of LPS-induced IL-10 secretion, indicating that PKC, once activated in vivo, might play a prominent role in controlling the secretion of IL-10 by AM. This study provides evidence that the PKC activator PMA and the phosphatase inhibitor calyculin A are able to dissociate TNF-alpha from IL-10 secretion by AM. Our data further indicate that LPS-mediated activation of certain signalling molecules has different consequences on the secretion of TNF-alpha or IL-10 by AM, an observation which may be important for the modulation of immune and inflammatory processes.     

The autocrine regulation of the proliferation of cell line A-549 under conditions facilitating the acidification of the culture medium]

The influence of serum-free media, previously conditioned by A-549 line cells of the human lung adenocarcinoma (c-medium), on the intensity of 3H-thymidine incorporation into DNA of the same cells was studied. It was found that, depending on the duration of conditioning (2, 4 and 6 days), the c-media were obtained with corresponding values of pH--7.2, 6.9 and 6.3, in the latter case the contact inhibition of cell growth being seen. On culturing the A-549 line cells in the c-medium at pH 7.2 and 6.9, the intensity of DNA biosynthesis was shown to be 2.4 and 1.8 times higher, respectively, compared to that under condition of the fresh serum-free medium. The cell culturing in the c-medium at pH 6.3 (here and in the case of pH 6.9 c-medium the media pH were made up to 7.2 before utilization) leads to the inhibition of DNA biosynthesis intensity in the cells. It was also detected that a temporary acidification of the pH 7.2 c-medium to pH 4.0 or 2.0, using, respectively CO2 bubbling or HCl titration, caused the growth inhibiting manifestation in this medium. The results obtained testify that the carcinoma cells of A-549 line are able to secrete into the cultured medium both stimulators and inhibitors of DNA biosynthesis, with a transforming growth factor beta being of primary importance among the latter.     

Evaluation of inflammatory cytokine secretion by human alveolar macrophages

The alveolar macrophage (AM) secretes interleukin 1beta (IL-1beta), tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and interleukin-8 (IL-8), all of them inflammatory cytokines involved in the pathogenesis of many lung diseases. The aim of the present work was to evaluate the basal and stimulated secretion of these cytokines by human AMs. Human AMs were collected by bronchoalveolar lavage (BAL) from four healthy controls and 13 patients with diffuse interstitial lung disease (five cases of sarcoidosis, three of hypersensitivity pneumonitis and five of idiopathic pulmonary fibrosis). AMs were cultured in the presence or absence of different concentrations of lipopolysaccharide (LPS), phorbolmyristate and gamma-interferon. IL-1beta, TNF-alpha, IL-6 and IL-8 levels were measured in BAL fluid and culture supernatant using specific enzyme-linked immunosorbent assays. The substance found to stimulate the secretion of inflammatory cytokines to the greatest extent was LPS at a concentration of 10 microg/ml. Regarding the secretion of IL-1beta, four observations were of interest: basal secretion was very low; LPS exerted a potent stimulatory effect; considerable within-group variability was observed; and there were no significant differences in the comparisons among groups. With respect to TNF-alpha secretion, the results were similar. The only striking finding was the higher basal secretion of this cytokine with respect to that of IL-1beta. Regarding the secretion of IL-6, the same pattern followed by TNF-alpha was found. However, it should be stressed that the increase induced by LPS was smaller than in the two previous cytokines. Regarding the secretion of IL-8, three findings were patent: the strong basal secretion of this cytokine; the moderate increase induced by LPS; and the existence of significant differences among the different groups with respect to the stimulated secretion of this cytokine, which reached maximum values in patients with idiopathic pulmonary fibrosis. Finally, it should be noted that the pattern of cytokines observed in the BAL fluid was similar to that found in cultured AM supernatants. The pattern of inflammatory cytokine secretion by AMs differs from that of other cells of the mononuclear phagocyte system (MPS). In this sense. AMs secrete low amounts of IL-1, moderate amounts of TNF-alpha and IL-6, and high quantities of IL-8. Adherence is an important stimulus in the secretion of these molecules and LPS elicits an increased secretion inverse to the basal secretion. There is considerable individual variability in the secretion of inflammatory cytokines by the AMs of patients with interstitial lung disease and the AMs of these patients are primed in vivo for the secretion of these cytokines. The results of our study, carried out in vitro, can be extrapolated to the in vivo setting.     

Adrenomedullin and adrenomedullin binding protein-1 downregulate TNF-alpha in macrophage cell line and rat Kupffer cells

Recent studies have demonstrated that administration of adrenomedullin (AM) and AM binding protein-1 (AMBP-1) maintains cardiovascular stability and reduces mortality in sepsis. However, the mechanism responsible for the beneficial effect of AM/AMBP-1 remains unknown. The aim of this study therefore was to determine whether AM/AMBP-1 directly reduces lipopolysaccharide (LPS)-induced secretion of TNF-alpha from murine macrophage-like cell line RAW 264.7 cells and Kupffer cells isolated from normal rats. TNF-alpha release and gene expression were determined by ELISA and RT-PCR, respectively. The results indicated that LPS increased TNF-alpha production from RAW cells by 38-63-fold in a dose- and time-dependent manner. Although incubation with AM or AMBP-1 alone inhibited LPS-induced TNF-alpha release by 14-22% and 13-22%, respectively, AM and AMBP-1 in combination significantly suppressed TNF-alpha production (by 24-35%). Moreover, the upregulated TNF-alpha mRNA by LPS stimulation was significantly reduced by AM/AMBP-1, but not by AM or AMBP-1 alone. In the Kupffer cells primary culture, AM or AMBP-1 alone inhibited LPS-induced TNF-alpha production by 52% and 44%, respectively. Co-culture with AM/AMBP-1 markedly reduced TNF-alpha production (by 90%). Moreover, AM or AMBP-1 alone decreased TNF-alpha mRNA expression by 41% and 36%, respectively, whereas the combination of AM/AMBP-1 decreased its expression by 63%. These results indicate that AM and AMBP-1 in combination effectively suppress LPS-induced TNF-alpha expression and release especially from primary cultured Kupffer cells, suggesting that the downregulatory effect of AM/AMBP-1 on proinflammatory cytokine TNF-alpha may represent a mechanism responsible for their beneficial effects in preventing inflammatory responses and tissue damage in sepsis.     

NALP-3 inflammasome silencing attenuates ceramide-induced transepithelial permeability

The hallmark of acute lung injury (ALI) is the influx of proinflammatory cytokines into lung tissue and alveolar permeability that ultimately leads to pulmonary edema. However, the mechanisms involved in inflammatory cytokine production and alveolar permeability are unclear. Recent studies suggest that excessive production of ceramide has clinical relevance as a mediator of pulmonary edema and ALI. Our earlier studies indicate that the activation of inflammasome promotes the processing and secretion of proinflammatory cytokines and causes alveolar permeability in ALI. However, the role of ceramide in inflammasome activation and the underlying mechanism in relation to alveolar permeability is not known. We hypothesized that ceramide activates the inflammasome and causes inflammatory cytokine production and alveolar epithelial permeability. To test this hypothesis, we analyzed the lung ceramide levels during hyperoxic ALI in mice. The effect of ceramide on activation of inflammasome and production of inflammatory cytokine was assessed in primary mouse alveolar macrophages and THP-1 cells. Alveolar transepithelial permeability was determined in alveolar epithelial type-II cells (AT-II) and THP-1 co-cultures. Our results reveal that ceramide causes inflammasome activation, induction of caspase-1, IL-1β cleavage, and release of proinflammatory cytokines. In addition, ceramide further induces alveolar epithelial permeability. Short-hairpin RNA silencing of inflammasome components abrogated ceramide-induced secretion of proinflammatory cytokines in vitro. Inflammasome silencing abolishes ceramide-induced alveolar epithelial permeability in AT-II. Collectively, our results demonstrate for the first time that ceramide-induced secretion of proinflammatory cytokines and alveolar epithelial permeability occurs though inflammasome activation.     

Differential regulation of cytokine release and leukocyte migration by lipopolysaccharide-stimulated primary human lung alveolar type II epithelial cells and macrophages

Bacterial colonization is a secondary feature of many lung disorders associated with elevated cytokine levels and increased leukocyte recruitment. We hypothesized that, alongside macrophages, the epithelium would be an important source of these mediators. We investigated the effect of LPS (0, 10, 100, and 1000 ng/ml LPS, up to 24 h) on primary human lung macrophages and alveolar type II epithelial cells (ATII; isolated from resected lung tissue). Although macrophages produced higher levels of the cytokines TNF-alpha and IL-1beta (p < 0.0001), ATII cells produced higher levels of chemokines MCP-1, IL-8, and growth-related oncogene alpha (p < 0.001), in a time- and concentration-dependent manner. Macrophage (but not ATII cell) responses to LPS required activation of ERK1/2 and p38 MAPK signaling cascades; phosphorylated ERK1/2 was constitutively up-regulated in ATII cells. Blocking Abs to TNF-alpha and IL-1beta during LPS exposure showed that ATII cell (not macrophage) MCP-1 release depended on the autocrine effects of IL-1beta and TNF-alpha (p < 0.003, 24 h). ATII cell release of IL-6 depended on autocrine effects of TNF-alpha (p < 0.006, 24 h). Macrophage IL-6 release was most effectively inhibited when both TNF-alpha and IL-1beta were blocked (p < 0.03, 24 h). Conditioned media from ATII cells stimulated more leukocyte migration in vitro than conditioned media from macrophages (p < 0.0002). These results show differential activation of cytokine and chemokine release by ATII cells and macrophages following LPS exposure. Activated alveolar epithelium is an important source of chemokines that orchestrate leukocyte migration to the peripheral lung; early release of TNF-alpha and IL-1beta by stimulated macrophages may contribute to alveolar epithelial cell activation and chemokine production.     

Effects of macrophage inducible nitric oxide synthase in murine septic lung injury

Inducible nitric oxide synthase (iNOS) contributes importantly to septic pulmonary protein leak in mice with septic acute lung injury (ALI). However, the role of alveolar macrophage (AM) iNOS in septic ALI is not known. Thus we assessed the specific effects of AM iNOS in murine septic ALI through selective AM depletion (via intratracheal instillation of clodronate liposomes) and subsequent AM reconstitution (via intratracheal instillation of donor iNOS+/+ or iNOS-/- AM). Sepsis was induced by cecal ligation and perforation, and ALI was assessed at 4 h: protein leak by the Evans blue (EB) dye method, neutrophil infiltration via myeloperoxidase (MPO) activity, and pulmonary iNOS mRNA expression via RT-PCR. In iNOS+/+ mice, AM depletion attenuated the sepsis-induced increases in pulmonary microvascular protein leak (0.3 +/- 0.1 vs. 1.4 +/- 0.1 microg EB.g lung(-1).min(-1); P < 0.05) and MPO activity (37 +/- 4 vs. 67 +/- 8 U/g lung; P < 0.05) compared with that shown in non-AM-depleted mice. In AM-depleted iNOS+/+ mice, septic pulmonary protein leak was restored by AM reconstitution with iNOS+/+ AM (0.9 +/- 0.3 microg EB.g lung(-1).min(-1)) but not with iNOS-/- donor AM. In iNOS-/- mice, sepsis did not induce pulmonary protein leak or iNOS mRNA expression, despite increased pulmonary MPO activity. However, AM depletion in iNOS-/- mice and subsequent reconstitution with iNOS+/+ donor AM resulted in significant sepsis-induced pulmonary protein leak and iNOS expression. Septic pulmonary MPO levels were similar in all AM-reconstituted groups. Thus septic pulmonary protein leak is absolutely dependent on the presence of functional AM and specifically on iNOS in AM. AM iNOS-dependent pulmonary protein leak was not mediated through changes in pulmonary neutrophil influx.     

Alveolar macrophage activation is a key initiation signal for acute lung ischemia-reperfusion injury

Lung ischemia-reperfusion (I/R) injury is a biphasic inflammatory process. Previous studies indicate that the later phase is neutrophil-dependent and that alveolar macrophages (AMs) likely contribute to the acute phase of lung I/R injury. However, the mechanism is unclear. AMs become activated and produce various cytokines and chemokines in many inflammatory responses, including transplantation. We hypothesize that AMs respond to I/R by producing key cytokines and chemokines and that depletion of AMs would reduce cytokine/chemokine expression and lung injury after I/R. To test this, using a buffer-perfused, isolated mouse lung model, we studied the impact of AM depletion by liposome-clodronate on I/R-induced lung dysfunction/injury and expression of cytokines/chemokines. I/R caused a significant increase in pulmonary artery pressure, wet-to-dry weight ratio, vascular permeability, tumor necrosis factor (TNF)-alpha, monocyte chemoattractant protein (MCP)-1, and macrophage inflammatory protein (MIP)-2 expression, as well as decreased pulmonary compliance, when compared with sham lungs. After AM depletion, the changes in each of these parameters between I/R and sham groups were significantly attenuated. Thus AM depletion protects the lungs from I/R-induced dysfunction and injury and significantly reduces cytokine/chemokine production. Protein expression of TNF-alpha and MCP-1 are positively correlated to I/R-induced lung injury, and AMs are a major producer/initiator of TNF-alpha, MCP-1, and MIP-2. We conclude that AMs are an essential player in the initiation of acute lung I/R injury.     

Mechanism of CO2 acquisition in an acid-tolerant Chlamydomonas

The acid-tolerant green alga Chlamydomonas (UTCC 121) grows in media ranging in pH from 2.5 to 7.0. Determination of the overall internal pH of the cells, using (14)C-benzoic acid (BA) or [2-(14)C]-5,5-dimethyloxazolidine-2,4-dione (DMO), showed that the cells maintain a neutral pH (6.6 to 7.2) over an external pH range of 3.0-7.0. The cells express an external carbonic anhydrase (CA) when grown in media above pH 5.5, and CA increases to a maximum at pH 7.0. Removal of external CA by trypsin digestion or by acetazolamide (AZA) inhibition indicated that CA was essential for photosynthesis at pH 7.0 and that the cells had no capacity for direct bicarbonate uptake. Monitoring of CO(2) uptake and O(2) evolution by mass spectrometry during photosynthesis did not provide any evidence of active CO(2) uptake. The CO(2) compensation concentration of the cells ranged from 9.4 microM at pH 4.5 to 16.2 microM at pH 7.0. An examination of the kinetics of ribulose 1.5-bisphosphate carboxylase/oxygenase (Rubisco), in homogenates of cells grown at pH 7.0, showed that the K(m) (CO(2)) was 16.3 microM. These data indicate that the pH between the cell interior and the external medium was large enough at acid pH to allow the accumulation of inorganic carbon (Ci) by the diffusive uptake of CO(2), and the expression of external CA at neutral pH values would maintain an equilibrium CO(2) concentration at the cell surface. This species does not possess a CO(2)-concentrating mechanism because the whole cell affinity for Ci appears to be determined by the low K(m) (CO(2)) Rubisco of the alga.     

Effect of ethanesulfonic acid buffers and pH on the accumulation of a nervous system-specific protein (S-100) and a glial-enriched enzyme in a clonal line of rat astrocytes (C6).

Stationary phase cultures of a clonal line of rat astrocytes (C6) were maintained at pH values ranging from 6.0 to 8.4 using media buffered with various combinations of organic buffers or graded concentrations of bicarbonate ion at a constant CO2 tension. The accumulation of a soluble acidic protein unique to the nervous system (S-100) in media buffered with organic buffers was optimal in the pH range 6.4 to 6.8, significantly more acid than that optimal for cell growth (pH 7.0 to 7.8). Cells maintained in CO2-bicarbonate-buffered media exhibited a higher and less marked pH optimum for S-100 protein accumulation and a lower efficiency of accumulation of the protein. These data suggest that the organic buffer ions themselves, apart from their function as buffers, are influencing the accumulation of S-100. The specific activity (assayed at the enzymatic pH optimum) of a membrane-bound enzyme enriched in glial cells and myelin, 2',3'-cyclic nucleotide 3'-phosphohydrolase, was markedly pH-dependent. The optimal pH range was 6.4 to 6.7 in organic buffer controlled media. In CO2-bicarbonate controlled media the optimal pH range was only slightly higher (pH 6.6 to 7.0), but the specific activities were reduced relative to organic buffer-grown cells. The structural relationship of some of the aminoethanesulfonic acid buffers used in these experiments to certain compounds of neurochemical interest (such as taurine and alpha-flupenthixol) is noted.     

Suppression of lncRNA NLRP3 inhibits NLRP3-triggered inflammatory responses in early acute lung injury

Acute lung injury (ALI) is a common lung pathology that is accompanied by alveolar macrophage (AM) activation and inflammatory response. This study investigated the role of the long non-coding RNA NONRATT004344 (hereafter named lncRNA NLRP3) in regulating the Nod-like receptor protein 3 (NLRP3)-triggered inflammatory response in early ALI and the underlying mechanism as well. We established LPS-induced ALI models to explore their interactive mechanisms in vitro and in vivo. Luciferase reporter assays were performed to determine that miR-138-5p could bind to lncRNA NLRP3 and NLRP3. We observed increased lncRNA NLRP3 expression, decreased miR-138-5p expression, NLRP3 inflammasome activation, and upregulated caspase-1, IL-1β, and IL-18 expression in the LPS-induced ALI model. Furthermore, lncRNA NLRP3 overexpression activated the NLRP3 inflammasome and promoted IL-1β and IL-18 secretion; the miR-138-5p mimic abolished these effects in vivo and in vitro. Consistently, miR-138-5p inhibition reversed the effects of lncRNA NLRP3 silencing on the expression of NLRP3-related molecules and inhibition of the NLRP3/caspase-1/IL-1β signalling pathway. Mechanistically, lncRNA NLRP3 sponging miR-138-5p facilitated NLRP3 activation through a competitive endogenous RNA (ceRNA) mechanism. In summary, our results suggested that lncRNA NLRP3 binding miR-138-5p promotes NLRP3-triggered inflammatory response via lncRNA NLRP3/miR-138-5p/NLRP3 ceRNA network (ceRNET) and provides insights into the treatment of early ALI.Subject terms: Bacterial infection, Inflammasome     

Chitosan oligosaccharide (COS) inhibits LPS-induced inflammatory effects in RAW 264.7 macrophage cells

The focus of this study was to clarify the relation between the nitric oxide (NO) production and cytokine expression including tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6), and also investigated the effect of COS on LPS stimuli from RAW 264.7 cell. The lipopolysaccharide (LPS) of Gram-negative bacteria induces the expression of cytokines and potent inducers of inflammatory cytokines such as TNF-alpha and IL-6. In this experiment, upon stimulation with increasing concentrations of chitosan, the LPS-stimulated TNF-alpha and IL-6 secretion was significantly recovered within the incubation media of RAW 264.7 cells. Consistently, RT-PCR with mRNA and Western blot with anti-cytokine antiserum including TNF-alpha and IL-6 showed that the amount of TNF-alpha and IL-6 secretion in the incubation media recovered with the concentration of chitosan. The LPS-stimulated NO secretion was significantly recovered within the 6h and 12h incubation media of RAW 264.7 cells, too. The recovery effect of chitosan on IL-6 and NO secretion may be induced via the stimulus of TNF-alpha in RAW 264.7 cell. These results once again suggest that chitosan oligosaccharide may have the anti-inflammatory effect via the stimulus of TNF-alpha in the LPS-stimulated inflammation in RAW 264.7 cells.     

Impaired functional activity of alveolar macrophages from GM-CSF-deficient mice

We hypothesized that pulmonary granulocyte-macrophage colony-stimulating factor (GM-CSF) is critically involved in determining the functional capabilities of alveolar macrophages (AM) for host defense. To test this hypothesis, cells were collected by lung lavage from GM-CSF mutant mice [GM(-/-)] and C57BL/6 wild-type mice. GM(-/-) mice yielded almost 4-fold more AM than wild-type mice. The percentage of cells positive for the beta(2)-integrins CD11a and CD11c was reduced significantly in GM(-/-) AM compared with wild-type cells, whereas expression of CD11b was similar in the two groups. The phagocytic activity of GM(-/-) AM for FITC-labeled microspheres was impaired significantly compared with that of wild-type AM both in vitro and in vivo (after intratracheal inoculation with FITC-labeled beads). Stimulated secretion of tumor necrosis factor-alpha (TNF-alpha) and leukotrienes by AM from the GM(-/-) mice was greatly reduced compared with wild-type AM, whereas secretion of monocyte chemoattractant protein-1 was increased. Transgenic expression of GM-CSF exclusively in the lungs of GM(-/-) mice resulted in AM with normal or supranormal expression of CD11a and CD11c, phagocytic activity, and TNF-alpha secretion. Thus, in the absence of GM-CSF, AM functional capabilities for host defense were significantly impaired but were restored by lung-specific expression of GM-CSF.     

Isovitexin Exerts Anti-Inflammatory and Anti-Oxidant Activities on Lipopolysaccharide-Induced Acute Lung Injury by Inhibiting MAPK and NF-κB and Activating HO-1/Nrf2 Pathways

Oxidative damage and inflammation are closely associated with the pathogenesis of acute lung injury (ALI). Thus, we explored the protective effect of isovitexin (IV), a glycosylflavonoid, in the context of ALI. To accomplish this, we created in vitro and in vivo models by respectively exposing macrophages to lipopolysaccharide (LPS) and using LPS to induce ALI in mice. In vitro, our results showed that IV treatment reduced LPS-induced pro-inflammatory cytokine secretion, iNOS and COX-2 expression and decreased the generation of ROS. Consistent findings were obtained in vivo. Additionally, IV inhibited H₂O₂-induced cytotoxicity and apoptosis. However, these effects were partially reversed following the use of an HO-1 inhibitor in vitro. Further studies revealed that IV significantly inhibited MAPK phosphorylation, reduced NF-κB nuclear translocation, and upregulated nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) expression in RAW 264.7 cells. In vivo, pretreatment with IV attenuated histopathological changes, infiltration of polymorphonuclear granulocytes and endothelial activation, decreased the expression of ICAM-1 and VCAM-1, reduced the levels of MPO and MDA, and increased the content of GSH and SOD in ALI. Furthermore, IV treatment effectively increased Nrf2 and HO-1 expression in lung tissues. Therefore, IV may offer a protective role against LPS-induced ALI by inhibiting MAPK and NF-κB and activating HO-1/Nrf2 pathways.     

Cardamonin inhibits LPS-induced inflammatory responses and prevents acute lung injury by targeting myeloid differentiation factor 2

BackgroundAcute lung injury (ALI) is a systemic inflammatory process, which has no pharmacological therapy in clinic. Accumulating evidence has demonstrated that natural compounds from herbs have potent anti-inflammatory efficacy in several disease models, which could be the potential candidates for the treatment of ALI.Hypothesis/PurposeAnti-inflammatory screening from natural product bank may provide new anti-inflammatory compounds for therapeutic target discovery and ALI treatment.Methods165 natural compounds were screened for their anti-inflammatory activity in LPS-stimulated macrophages. PCR array, SPR and ELISA were used to determine the potential target of the most active compound, Cardamonin (CAR). The pharmacological effect of CAR was further evaluated in both LPS-stimulated macrophages and ALI mice model.ResultsOut of the screened 165 compounds, CAR significantly inhibited LPS-induced inflammatory cytokine secretion in macrophages. We further showed that CAR significantly inhibited NF-κB and JNK signaling activation, and thereby inflammatory cytokine production via directly interacting with MD2 in vitro. In vivo, our data show that CAR treatment inhibited LPS-induced lung damage, systemic inflammatory cytokine production, and reduced macrophage infiltration in the lungs, accompanied with reduced TLR4/MD2 complex in lung tissues, Treatment with CAR also dose-dependently increased survival in the septic mice induced by DH5α bacterial infection.ConclusionWe demonstrate that a natural product, CAR, attenuates LPS-induced lung injury and sepsis by inhibiting inflammation via interacting with MD2, leading to the inactivation of the TLR4/MD2-MyD88-MAPK/NF-κB pathway.     

Beneficial effects of ApoA-I on LPS-induced acute lung injury and endotoxemia in mice

High density lipoprotein (HDL) binds lipopolysaccharide (LPS) and neutralizes its toxicity. The aim of our study was to investigate the effects of Apolipoprotein (ApoA-I), the major apolipoprotein of HDL, on LPS-induced acute lung injury (ALI) and endotoxemia. BALB/c mice were challenged with LPS, followed by ApoA-I or saline administration for 24h. The mice were then sacrificed and histopathological analysis of the lung was performed. We found that ApoA-I could attenuate LPS-induced acute lung injury and inflammation. To investigate the mechanisms, we measured tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) levels in the serum and bronchoalveolar lavage (BAL) fluid and found that ApoA-I could significantly inhibit LPS-induced increases in the IL-1beta and TNF-alpha levels in serum (P<0.05, respectively), as well as in the IL-1beta, TNF-alpha, and IL-6 levels in BAL fluid (P<0.01 and P<0.05, P<0.05, respectively). Moreover, we evaluated the effect of ApoA-I on the mortality of L-929 cells which were attacked by LPS-activated peritoneal macrophages. We found that ApoA-I could significantly inhibit the LPS-induced cell death in a dose-dependent fashion. Furthermore, we investigated in vivo the effects of ApoA-I on the mortality rate and survival time after LPS administration and found that ApoA-I significantly decreased the mortality (P<0.05) and increased the survival time (P<0.05). In summary, the results suggest that ApoA-I could effectively protect against LPS-induced endotoxemia and acute lung damage. The mechanism might be related to inhibition of inflammatory cytokine release from macrophages.