Pulmonary Surfactant Phosphatidylglycerol Inhibits Mycoplasma pneumoniae-stimulated Eicosanoid Production from Human and Mouse Macrophages
Department of Medicine, National Jewish Health, Denver, Colorado, 80206, USA. Journal of Biological Chemistry
(Impact Factor: 4.57).
03/2011; 286(10):7841-53. DOI: 10.1074/jbc.M110.170241
Mycoplasma pneumoniae is a human pathogen causing respiratory infections that are also associated with serious exacerbations of chronic lung diseases.
Membranes and lipoproteins from M. pneumoniae induced a 4-fold increase in arachidonic acid (AA) release from RAW264.7 and a 2-fold increase in AA release from primary
human alveolar macrophages. The bacterial lipoprotein mimic and TLR2/1 agonist Pam3Cys and the TLR2/6 agonist MALP-2 produced
effects similar to those elicited by M. pneumoniae in macrophages by inducing the phosphorylation of p38MAPK and p44/42ERK1/2 MAP kinases and cyclooxygenase-2 (COX-2) expression. M. pneumoniae induced the generation of prostaglandins PGD2 and PGE2 from RAW264.7 cells and thromboxane B2 (TXB2) from human alveolar macrophages. Anti-TLR2 antibody completely abolished M. pneumoniae-induced AA release and TNFα secretion from RAW264.7 cells and human alveolar macrophages. Disruption of the phosphorylation
of p44/42ERK1/2 or inactivation of cytosolic phospholipase A2α (cPLA2α) completely inhibited M. pneumoniae-induced AA release from macrophages. The minor pulmonary surfactant phospholipid, palmitoyl-oleoyl-phosphatidylglycerol (POPG),
antagonized the proinflammatory actions of M. pneumoniae, Pam3Cys, and MALP-2 by reducing the production of AA metabolites from macrophages. The effect of POPG was specific, insofar
as saturated PG, and saturated and unsaturated phosphatidylcholines did not have significant effect on M. pneumoniae-induced AA release. Collectively, these data demonstrate that M. pneumoniae stimulates the production of eicosanoids from macrophages through TLR2, and POPG suppresses this pathogen-induced response.
Available from: Markus Fehrholz
- "The latter results are in accordance with previous data from our group and with results from a number of other studies on natural surfactants such as Curo- surf1[54,56,58,62,63,65,686970. Although described in previous studies[64,67,74], we did not find an equivalent anti-inflammatory activity when evaluating the major synthetic components of CHF5633 alone. As far as anti-inflammatory IL-10 is concerned, we found a slightly, but not significantly enhanced mRNA but not protein expression in LPS-stimulated monocytes following 14h exposure to both CHF5633 and Curosurf1. "
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Surfactant replacement therapy is the standard of care for the prevention and treatment of neonatal respiratory distress syndrome. New generation synthetic surfactants represent a promising alternative to animal-derived surfactants. CHF5633, a new generation reconstituted synthetic surfactant containing SP-B and SP-C analogs and two synthetic phospholipids has demonstrated biophysical effectiveness in vitro and in vivo. While several surfactant preparations have previously been ascribed immunomodulatory capacities, in vitro data on immunomodulation by CHF5633 are limited, so far. Our study aimed to investigate pro- and anti-inflammatory effects of CHF5633 on native and LPS-stimulated human adult monocytes.
Highly purified adult CD14+ cells, either native or simultaneously stimulated with LPS, were exposed to CHF5633, its components, or poractant alfa (Curosurf®). Subsequent expression of TNF-α, IL-1β, IL-8 and IL-10 mRNA was quantified by real-time quantitative PCR, corresponding intracellular cytokine synthesis was analyzed by flow cytometry. Potential effects on TLR2 and TLR4 mRNA and protein expression were monitored by qPCR and flow cytometry.
Neither CHF5633 nor any of its components induced inflammation or apoptosis in native adult CD14+ monocytes. Moreover, LPS-induced pro-inflammatory responses were not aggravated by simultaneous exposure of monocytes to CHF5633 or its components. In LPS-stimulated monocytes, exposure to CHF5633 led to a significant decrease in TNF-α mRNA (0.57 ± 0.23-fold, p = 0.043 at 4h; 0.56 ± 0.27-fold, p = 0.042 at 14h). Reduction of LPS-induced IL-1β mRNA expression was not significant (0.73 ± 0.16, p = 0.17 at 4h). LPS-induced IL-8 and IL-10 mRNA and protein expression were unaffected by CHF5633. For all cytokines, the observed CHF5633 effects paralleled a Curosurf®-induced modulation of cytokine response. TLR2 and TLR4 mRNA and protein expression were not affected by CHF5633 and Curosurf®, neither in native nor in LPS-stimulated adult monocytes.
The new generation reconstituted synthetic surfactant CHF5633 was tested for potential immunomodulation on native and LPS-activated adult human monocytes. Our data confirm that CHF5633 does not exert unintended pro-inflammatory effects in both settings. On the contrary, CHF5633 significantly suppressed TNF-α mRNA expression in LPS-stimulated adult monocytes, indicating potential anti-inflammatory effects.
Available from: Robert Podolsky
- "Pattern recognition receptors can be activated not only in reponse to pathogen-associated molecular patterns but also damage-associated molecular patterns arising from cell injury (reviewed in , ). For example, PG, produced by alveolar cells as a significant component of pulmonary surfactant, inhibits TLR signaling in macrophages exposed to LPS in vitro, acting at multiple sites to disrupt TLR4 signaling , as well as TLR2 pathway activation in response to bacterial and mycoplasma byproducts in vitro
. PG also protects the lungs from inflammation initiated by LPS exposure in vivo
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ABSTRACT: We have previously shown that liposomes composed of egg-derived phosphatidylglycerol (PG), with a mixed fatty acid composition (comprising mainly palmitate and oleate), inhibit the proliferation and promote the differentiation of rapidly dividing keratinocytes, and stimulate the growth of slowly proliferating epidermal cells. To determine the species of PG most effective at modulating keratinocyte proliferation, primary mouse keratinocytes were treated with different PG species, and proliferation was measured. PG species containing polyunsaturated fatty acids were effective at inhibiting rapidly proliferating keratinocytes, whereas PG species with monounsaturated fatty acids were effective at promoting proliferation in slowly dividing cells. Thus, palmitoyl-arachidonyl-PG (16∶0/20∶4), palmitoyl-linoleoyl-PG (16∶0/18∶2), dilinoleoyl-PG (18∶2/18∶2) and soy PG (a PG mixture with a large percentage of polyunsaturated fatty acids) were particularly effective at inhibiting proliferation in rapidly dividing keratinocytes. Conversely, palmitoyl-oleoyl-PG (16∶0/18∶1) and dioleoyl-PG (18∶1/18∶1) were especially effective proproliferative PG species. This result represents the first demonstration of opposite effects of different species of a single class of phospholipid and suggests that these different PG species may signal to diverse effector enzymes to differentially affect keratinocyte proliferation and normalize keratinocyte proliferation. Thus, different PG species may be useful for treating skin diseases characterized by excessive or insufficient proliferation.
Available from: PubMed Central
- "In this sense, there must be some intrinsic or adaptive mechanisms to protect against dysfunctional inflammation during infection. An increasing number of reports have indicated that the hosts have developed sophisticated negative mechanism to regulate the multiple layers of inflammatory response , , . Indeed, some cell wall components of bacteria, such as lipoteichoic acids and lipopolysaccharides, were reported to activate the basic leucine zipper transcription factor NF-E2-related factor 2 (Nrf2), a key factor involved in antioxidant protein expression in human tracheal smooth muscle cells and monocytes . "
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This study is to investigate the mechanisms by which macrophage-activating lipopeptide-2 (MALP-2) induces heme oxygenase (HO)-1, a cytoprotective enzyme that catalyzes the degradation of heme, in human monocytes.
Human monocytic THP-1 cells were cultured for transient transfection with plasmids and stimulation with MALP-2 for indicative time intervals. After incubation with MALP-2, cells were collected and disrupted, before being tested for promoter activity using luciferase assay. For analysis of proteins, immunoreactive bands were detected using an enhanced chemiluminescence Western blotting system, and the band intensity was measured by densitometryic analysis. For the detection of co-immunoprecipitation, SDS-PAGE was performed and the membranes were probed using respective antibodies. To investigate the cellular localization of NF-E2-related factor 2 (Nrf2), cells underwent immunofluorescence staining and confocal microscopy, and were analyzed using electrophoretic mobility shift assay.
MALP-2-induced HO-1 expression and promoter activity were abrogated by transfection with dominant negative (DN) plasmids of TLR2 and TLR6, or their neutralizing antibodies. However, inhibition of MyD88 or transfection with the DN-MyD88 was insufficient to attenuate HO-1 expression. In contrast, mutation or silencing of MyD88 adapter-like (Mal) by DN-Mal or siRNA almost completely blocked HO-1 induction. Btk, c-Src and PI3K were also involved in MALP-2-induced HO-1 expression, as revealed by specific inhibitors LFM-A13, PP1 and LY294002, or by transfection with siRNA of c-Src. MALP-2-induced activation of PI3K was attenuated by transfection with DN mutant of Mal, and by pretreatment with LFM-A13 or PP1. Furthermore, MALP-2 stimulated the translocation of Nrf2 from the cytosol to the nucleus and Nrf2 binding to the ARE site in the HO-1 promoter, which could also be inhibited by pretreatment with a PI3K inhibitor, LY294002.
These results indicated that MALP-2 required TLR2/6, Btk, Mal and c-Src to activate PI3K, which in turn initiated the activation of Nrf2 for efficient HO-1 induction.
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