Anti-inflammatory and antibacterial activities of Pyrrosia petiolosa ethyl acetate (PPEAE) against Staphylococcus aureus in mice.

P. petiolosa as a typical Chinese herbal medicine has been generally utilized as Chinese native medicine formulation for treatment of chronic bronchitis, bronchial asthma and pneumoconiosis. The objective of this study was to evaluate the anti-inflammatory and antibacterial activities of P. petiolosa ethyl acetate extract (PPEAE) against S. aureusin mice. In our study, mice were infected pneumonia by S. aureus, colonization of S. aureus in lung tissue was calculated and the number of white blood cells (WBC) in blood was measured. Meanwhile, the hematoxylin-eosin staining (H&E) was observed and the Real-time PCR was employed to determine the relative mRNA expression. The results showed that, after treated with PPEAE the wet/dry (W/D) weight ratio and the number of WBC decreased dramatically, the number of S. aureus was significantly reduced. Furthermore, H&E staining showed that PPEAE obviously relieved the inflammation of infected mice and real-time PCR results indicated that PPEAE significantly down regulated the inflammatory iNOS, TNF-α and up regulated the anti-inflammatory HO-1 mRNA. In summary, our study revealed that application of crude product PPEAE had prominent antibacterial activity against S. aureus. PPEAE significantly reduced the biomass of S. aureus and effectively relieved the inflammation of S. aureus-induced pneumonia.

SHP2 deficiency promotes Staphylococcus aureus pneumonia following influenza infection.

OBJECTIVES: Secondary bacterial pneumonia is common following influenza infection. However, it remains unclear about the underlying molecular mechanisms. MATERIALS AND METHODS: We established a mouse model of post-influenza S aureus pneumonia using conditional Shp2 knockout mice (LysMCre/+ :Shp2flox/flox ). The survival, bacterial clearance, pulmonary histology, phenotype of macrophages, and expression of type I interferons and chemokines were assessed between SHP2 deletion and control mice (Shp2flox/flox ). We infused additional KC and MIP-2 to examine the reconstitution of antibacterial immune response in LysMCre/+ :Shp2flox/flox mice. The effect of SHP2 on signal molecules including MAPKs (JNK, p38 and Erk1/2), NF-κB p65 and IRF3 was further detected. RESULTS: LysMCre/+ :Shp2flox/flox mice displayed impaired antibacterial immunity and high mortality compared with control mice in post-influenza S aureus pneumonia. The attenuated antibacterial ability was associated with the induction of type I interferon and suppression of chemo-attractants KC and MIP-2, which reduced the infiltration of neutrophils into the lung upon secondary bacterial invasion. In additional, Shp2 knockout mice displayed enhanced polarization to alternatively activated macrophages (M2 phenotype). Further in vitro analyses consistently demonstrated that SHP2-deficient macrophages were skewed towards an M2 phenotype and had a decreased antibacterial capacity. Moreover, SHP2 modulated the inflammatory response to secondary bacterial infection via interfering with NF-κB and IRF3 signalling in macrophages. CONCLUSIONS: Our findings reveal that the SHP2 expression enhances the host immune response and prompts bacterial clearance in post-influenza S aureus pneumonia.

miR-182-5p contributes to intestinal injury in a murine model of Staphylococcus aureus pneumonia-induced sepsis via targeting surfactant protein D.

Sepsis is a severe clinical disease, which is resulted from the excessive host inflammation response to the infection. Growing evidence indicates that Staphylococcus aureus pneumonia is a significant cause of sepsis, which can lead to intestinal injury, inflammation, and apoptosis. Studies have shown that miR-182-5p can serve as a tumor oncogene or a tumor suppressive microRNA in various cancers, however, its biological role in sepsis is still uninvestigated. Here, we reported that miR-182-5p was obviously increased in S. aureus pneumonia mice models. Loss of miR-182-5p inhibited intestinal damage and intestinal apoptosis as indicated by the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. In addition, we observed the lack of miR-182-5p altered the local inflammatory response to pneumonia in the intestine. Elevated tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels were observed in intestinal tissue of pneumonia groups compared with the shams. Furthermore, miR-182-5p knockout (KO) pneumonia group demonstrated decreased levels of intestinal TNF-α and IL-6. Primary murine intestinal epithelial cells were isolated and cultured in our investigation. We exhibited downregulation of miR-182-5p repressed intestinal epithelial cells apoptosis and rescued the cell viability. Meanwhile, miR-182-5p caused elevated cell apoptosis and reduced cell proliferation. Moreover, the surfactant protein D (SP-D) binds with the bacterial pathogens and remove the pathogens and apoptotic bodies, which exhibits important roles in modulating immune responses. It was displayed in our study that SP-D was greatly decreased in pneumonia mice models. SP-D was predicted as a downstream target of miR-182-5p. These data concluded that miR-182-5p promoted intestinal injury in S. aureus pneumonia-induced sepsis via targeting SP-D.

THE ANTIBACTERIAL EFFECT OF URENA LOBATA L. FROMV GUANGXI ON MICE WITH STAPHYLOCOCCUS AUREUS PNEUMONIA.

BACKGROUND: Alcohol extract from the root of Urena lobata L. (ULL) had broad spectrum antimicrobial activity. Studies in vitro have sho that ULL aqueous extract has antibacterial effect on S. aureusis, and the combination therapy of the ULL aqueous extract with cefazolin sodium showed additive effect. MATERIALS AND METHODS: The mice underwent nasal inhalation with S. aureus, a subset of mice were intra-gastric gavage with ULL and/or intravenous injection cefazolin sodium twice daily. After being exposed to S. aureus for 5 days, 10 days and 14 days respectively, the white blood cells count (WBC), neutrophils absolute value (NEU) and the neutrophil percentage (NEU%) in peripheral blood, as well as the levels of serum immunoglobulin (Ig) G and IgM were determined using commercial kits. The colony count of S. aureus, the levels of interleukin (IL) -6 and IL-10 of mice lung tissue were detected, and the pathological changes of lung tissue were examined using H & E staining. RESULTS: ULL significantly protected against S. aureus pneumonia, as evidenced by the remarkable decrease in the rate of S. aureus colony count/lung weight, WBC, NEU and NEU% in peripheral blood, as well as the attenuation of lung histopathological damage. Additionally, ULL+cefazolin could have markedly reduced the rate of S. aureus colony count/lung weight when compared with cefazolin. Furthermore, ULL and ULL+cefazolin both could significantly decrease the serum levels of IgG and IgM, and the levels of IL-6, IL-10 in mice lung tissue. CONCLUSION: This study first demonstrated that ULL may have potential use as a therapeutic agent for S. aureus pneumonia, and the roles of IgG, IgM, IL-6 and IL-10 in ULL protection against S. aureus pneumonia remain to be further studied.

Lentiviral Vector Gene Therapy Protects XCGD Mice From Acute Staphylococcus aureus Pneumonia and Inflammatory Response.

Chronic granulomatous disease (CGD) is a primary immunodeficiency due to a deficiency in one of the subunits of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. CGD patients are characterized by an increased susceptibility to bacterial and fungal infections, and to granuloma formation due to the excessive inflammatory responses. Several gene therapy approaches with lentiviral vectors have been proposed but there is a lack of in vivo data on the ability to control infections and inflammation. We set up a mouse model of acute infection that closely mimic the airway infection in CGD patients. It involved an intratracheal injection of a methicillin-sensitive reference strain of S. aureus. Gene therapy, with hematopoietic stem cells transduced with regulated lentiviral vectors, restored the functional activity of NADPH oxidase complex (with 20-98% of dihydrorhodamine positive granulocytes and monocytes) and saved mice from death caused by S. aureus, significantly reducing the bacterial load and lung damage, similarly to WT mice even at low vector copy number. When challenged, gene therapy-treated XCGD mice showed correction of proinflammatory cytokines and chemokine imbalance at levels that were comparable to WT. Examined together, our results support the clinical development of gene therapy protocols using lentiviral vectors for the protection against infections and inflammation.

A calcium-redox feedback loop controls human monocyte immune responses: The role of ORAI Ca2+ channels.

In phagocytes, pathogen recognition is followed by Ca(2+) mobilization and NADPH oxidase 2 (NOX2)-mediated "oxidative burst," which involves the rapid production of large amounts of reactive oxygen species (ROS). We showed that ORAI Ca(2+) channels control store-operated Ca(2+) entry, ROS production, and bacterial killing in primary human monocytes. ROS inactivate ORAI channels that lack an ORAI3 subunit. Staphylococcal infection of mice reduced the expression of the gene encoding the redox-sensitive Orai1 and increased the expression of the gene encoding the redox-insensitive Orai3 in the lungs or in bronchoalveolar lavages. A similar switch from ORAI1 to ORAI3 occurred in primary human monocytes exposed to bacterial peptides in culture. These alterations in ORAI1 and ORAI3 abundance shifted the channel assembly toward a more redox-insensitive configuration. Accordingly, silencing ORAI3 increased the redox sensitivity of the channel and enhanced oxidation-induced inhibition of NOX2. We generated a mathematical model that predicted additional features of the Ca(2+)-redox interplay. Our results identified the ORAI-NOX2 feedback loop as a determinant of monocyte immune responses.

Surfactant Proteins SP-A and SP-D Ameliorate Pneumonia Severity and Intestinal Injury in a Murine Model of Staphylococcus Aureus Pneumonia.

UNLABELLED: Staphylococcus aureus pneumonia is an important cause of sepsis which causes gut injury, inflammation, and apoptosis. The surfactant proteins surfactant protein A (SP-A) and surfactant protein D (SP-D) bind bacterial pathogens and facilitate clearance of pathogens, apoptotic bodies, and modulate immune responses. SP-A and SP-D are expressed in both lung and gut epithelia. We hypothesize SP-A and SP-D regulate pneumonia severity and gut injury during pneumonia. METHODS: Wild-type (WT) and SP-A and SP-D double knockout (SP-A/D KO) mice were subjected to S. aureus or sham pneumonia. Bronchoalveolar lavage and tissue harvest were performed 24 h later. Pneumonia severity, gut mucosal injury, inflammation, and apoptosis were measured using a combination of histology, immunohistochemistry, cytokine assay, TUNEL assay, quantitative real-time polymerase chain reaction, and Western blot analyses. RESULTS: Pneumonia increased gut inflammation, apoptosis, and mucosal injury in both groups. Pneumonia histology and bacterial growth in bronchoalveolar lavage fluid demonstrate more severe infection in SP-A/D KO mice compared with WT controls. SP-A/D KO mice with pneumonia also demonstrate more severe histologic gut mucosal injury, increased gut apoptosis, elevated caspase-3 levels, and Bax/Bcl-2 mRNA expression compared with WT pneumonia mice. Nuclear factor κB (NF-κB) p65 expression and its nuclear translocation, gut levels of tumor necrosis factor α and interleukin-1ß were all increased in SP-A/D KO mice with pneumonia compared with WT controls. CONCLUSIONS: These data provide evidence SP-A and SP-D attenuate S. aureus pneumonia severity resulting in decreased intestinal mucosal injury, apoptosis, and inflammation. Improved pulmonary clearance of S. aureus decreased caspase-3 and Bax/Bcl-2 expressions and decreased activation of the NF-κB signaling pathway in intestine represent potential mechanisms for the effects of SP-A and SP-D on gut injury during pneumonia.

DIFFERENTIAL SUSCEPTIBILITY OF HUMAN SP-B GENETIC VARIANTS ON LUNG INJURY CAUSED BY BACTERIAL PNEUMONIA AND THE EFFECT OF A CHEMICALLY MODIFIED CURCUMIN.

Staphylococcus aureus is a common cause of nosocomial pneumonia frequently resulting in acute respiratory distress syndrome (ARDS). Surfactant protein B (SP-B) gene expresses two proteins involved in lowering surface tension and host defense. Genotyping studies demonstrate a significant association between human SP-B genetic variants and ARDS. Curcumins have been shown to attenuate host inflammation in many sepsis models. Our hypothesis is that functional differences of SP-B variants and treatment with curcumin (CMC2.24) modulate lung injury in bacterial pneumonia. Humanized transgenic mice, expressing either SP-B T or C allele without mouse SP-B gene, were used. Bioluminescent labeled S. aureus Xen 36 (50 µL) was injected intratracheally to cause pneumonia. Infected mice received daily CMC2.24 (40 mg/kg) or vehicle alone by oral gavage. Dynamic changes of bacteria were monitored using in vivo imaging system. Histological, cellular, and molecular indices of lung injury were studied in infected mice 48 h after infection. In vivo imaging analysis revealed total flux (bacterial number) was higher in the lung of infected SP-B-C mice compared with infected SP-B-T mice (P < 0.05). Infected SP-B-C mice demonstrated increased mortality, lung injury, apoptosis, and NF-κB expression compared with infected SP-B-T mice. Compared with controls, CMC2.24 treatment significantly reduced the following: mortality, total bacterial flux and lung tissue apoptosis, inflammatory cells, NF-κB expression (P < 0.05), and MMPs-2, -9, -12 activities (P < 0.05). We conclude that mice with SP-B-C allele are more susceptible to S. aureus pneumonia than mice with SP-B-T allele, and that CMC2.24 attenuates lung injury thus reducing mortality.

Staphylococcus aureus hemolysin A disrupts cell-matrix adhesions in human airway epithelial cells.

Treatment of primary or immortalized human airway epithelial cells (16HBE14o-, S9) or alveolar cancer cells (A549) with recombinant hemolysin A (rHla), a major virulence-associated factor of Staphylococcus aureus, induces alterations in cell shape and formation of paracellular gaps in the cell layer. Semiquantitative Western blotting using extracts of freshly isolated airway tissue (nasal epithelium) or 16HBE14o- model cells revealed that phosphorylation levels of focal adhesion kinase (Fak) and paxillin were altered upon treatment of tissue or cells with rHla. Immune fluorescence analyses showed that rHla treatment of 16HBE14o- cells results in losses of vinculin and paxillin from focal contacts and a net reduction in the number of focal contacts. The actin cytoskeleton was strongly remodeled. We concluded that treatment of cells with rHla activates Fak signaling, which accelerates focal contact turnover and prevents newly formed focal contacts (focal complexes) from maturation to focal adhesions. The inability of rHla-treated cells to form stable focal adhesions may be one factor that contributes to gap formation in the cell layer. In vivo, such changes may disturb the defensive barrier function of the airway epithelium and may facilitate lung infections by S. aureus.

Secretion of IL-16 through TNFR1 and calpain-caspase signaling contributes to MRSA pneumonia.

Staphylococcus aureus is a major cause of severe pneumonia. Multiple mechanisms of proinflammatory signaling are activated to recruit immune cells into the airway in response to S. aureus. We found that interleukin-16 (IL-16), a T cell cytokine that binds CD4, is potently activated by S. aureus, specifically by protein A (SpA), and to a much greater extent than by Gram-negative pathogens or lipopolysaccharide. IL-16 production involved multiple signals including ligation of tumor necrosis factor receptor (TNFR) family members or epidermal growth factor receptor, both receptors for SpA and generation of Ca(2+) fluxes to activate calpains and caspase-3. Although human airway epithelial cells, vascular endothelial cells, THP-1 and Jurkat T cells released IL-16 in response to S. aureus in vitro, in a murine model of pneumonia, CD4(+) cells were the major source of IL-16 suggesting the involvement of an autocrine signaling pathway. The production of IL-16 contributed to lung damage as neutralization of IL-16 enhanced S. aureus clearance and resulted in diminished lung pathology in S. aureus pneumonia. Our results suggest that the ability of S. aureus to activate TNFR1 and Ca(2+)/calpain signaling contribute to T cell activation and excessive inflammation in the setting of acute pneumonia.

Bacterial pathogens activate a common inflammatory pathway through IFNλ regulation of PDCD4.

The type III interferon (IFNλ) receptor IL-28R is abundantly expressed in the respiratory tract and has been shown essential for host defense against some viral pathogens, however no data are available concerning its role in the innate immune response to bacterial pathogens. Staphylococcus aureus and Pseudomonas aeruginosa induced significant production of IFNλ in the lung, and clearance of these bacteria from the lung was significantly increased in IL-28R null mice compared to controls. Improved bacterial clearance correlated with reduced lung pathology and a reduced ratio of pro- vs anti-inflammatory cytokines in the airway. In human epithelial cells IFNλ inhibited miR-21 via STAT3 resulting in upregulation of PDCD4, a protein known to promote inflammatory signaling. In vivo 18 hours following infection with either pathogen, miR-21 was significantly reduced and PDCD4 increased in the lungs of wild type compared to IL-28R null mice. Infection of PDCD4 null mice with USA300 resulted in improved clearance, reduced pathology, and reduced inflammatory cytokine production. These data suggest that during bacterial pneumonia IFNλ promotes inflammation by inhibiting miR-21 regulation of PDCD4.

Staphylococcus aureus protein A mediates invasion across airway epithelial cells through activation of RhoA GTPase signaling and proteolytic activity.

Staphyococcus aureus and especially the epidemic methicillin-resistant S. aureus strains cause severe necrotizing pneumonia. The mechanisms whereby these organisms invade across the mucosal epithelial barrier to initiate invasive infection are not well understood. Protein A (SpA), a highly conserved and abundant surface protein of S. aureus, activates TNF receptor 1 and EGF receptor (EGFR) signaling cascades that can perturb the cytoskeleton. We demonstrate that wild-type S. aureus, but not spa mutants, invade across polarized airway epithelial cell monolayers via the paracellular junctions. SpA stimulated a RhoA/ROCK/MLC cascade, resulting in the contraction of the cytoskeleton. SpA(+) but not SpA(-) mutants stimulated activation of EGFR and along with subsequent calpain activity cleaved the membrane-spanning junctional proteins occludin and E-cadherin, facilitating staphylococcal transmigration through the cell-cell junctions. Treatment of polarized human airway epithelial monolayers with inhibitors of ROCK, EGFR, MAPKs, or calpain prevented staphylococcal penetration through the monolayers. In vivo, blocking calpain activity impeded bacterial invasion into the lung parenchyma. Thus, S. aureus exploits multiple receptors available on the airway mucosal surface to facilitate invasion across epithelial barriers.

Hyper-IgE syndrome with STAT3 mutation: a case report in Mainland China.

Hyper-immunoglobulin E syndromes (HIES) including compound primary immunodeficiency and nonimmunological abnormalities are characterized by extremely high serum IgE levels, eosinophilia, eczema, susceptibility to infections, distinctive facial appearance, retention of deciduous teeth, cyst-forming pneumonias, and skeletal abnormalities. Itis reported that some cases of familial HIES are relative to autosomal dominant or recessive inheritance, but most cases are sporadic, and result from mutations in the human signal transducer and activator of transcription 3 (STAT3) gene. In this paper, we firstly report a young man diagnosed of Hyper-IgE syndrome with STAT3 mutation in Mainland China, and investigate the autosomal dominant trait of his family members.

[Effect of adenosine on activity of transcription factor NF-kappa B and cytokines in myocardial tissue of experimental rats with pneumonia].

OBJECTIVE: Recent studies have shown that cytokines TNF-alpha and IL-6 play important roles in myocardial injury or dysfunction. Transcription nuclear factor (NF-kappa B) have been implicated in the regulation of a variety of cytokines in response to cellular defense. The authors observed the activity of NF-kappa B and cytokines TNF-alpha, IL-6 mRNA expression in myocardium to further investigate the mechanism of myocardial injury caused by infectious pneumonia. The therapeutic effect of exogenous adenosine was also studied by observing the influence on NF-kappa B and cytokines. METHODS: Thirty rats were divided into three experimental groups at random, each group had 10 rats. The model of pneumonia was induced by the injection of Staphylococcus aureus into the trachea of rats. Adenosine-treated rats were given daily slow intravenous injection of adenosine at a dose of 150 microg/kg.min for 3 days from the second day. All rats were killed on the fifth day. Myocardial tissues were preserved in liquid nitrogen for examination. Pathological examination of myocardium was done and TNF-alpha and IL-6 mRNA expression was detected by reverse transcription polymerase chain reaction (RT-PCR). NF-kappa B activity was measured by electrophoretic mobility shift assay (EMSA). RESULTS: (1) The myocardium in pneumonia group showed significant pathological lesion when compared with control group (P < 0.01). The pathological lesion of myocardium in adenosine-treated group significantly decreased when compared to pneumonia group (P < 0.05). (2) Significant increase of TNF-alpha and IL-6 mRNA expression was observed in myocardium of pneumonic rats when compared with control group (2.27 +/- 0.27 vs. 1.05 +/- 0.16; 1.89 +/- 0.31 vs. 1.12 +/- 0.25: P < 0.01, respectively). NF-kappa B activity of myocardium in pneumonia group was significantly higher than that in control group (13,033 +/- 1286 vs. 383 +/- 15: P < 0.01). (3) TNF-alpha and IL-6 mRNA expression was significantly decreased in adenosine-treated group when compared with pneumonia group (1.25 +/- 0.18 vs. 2.27; 1.31 +/- 0.25 vs. 1.89 +/- 0.31, P < 0.01, respectively). Comparing to that in pneumonia group, NF-kappa B activity of myocardium in adenosine-treated group was significantly decreased (4 487 +/- 562 vs. 13033 +/- 1286, P < 0.01), but it was still significantly higher than that in control group (4487 +/- 562 vs.383 +/- 15, P < 0.01). CONCLUSIONS: Increased activity of NF-kappa B and subsequent upregulation of TNF-alpha and IL-6 mRNA expression probably play a pivotal role in the mechanism of myocardial injury in rats with pneumonia. Exogenous adenosine can inhibit inflammatory change by lowering NF-kappa B activity and subsequent down-regulation of TNF-alpha and IL-6 expression. Our findings provide novel therapeutic evidence of adenosine in myocardial injury induced by pneumonia in clinic.