Lung mRNA Research Articles

Vasoactive intestinal peptide overexpression mediated by lentivirus attenuates lipopolysaccharide-induced acute lung injury in mice by inhibiting inflammation

Vasoactive intestinal peptide (VIP) is one of the most abundant neuropeptides in the lungs with various biological characters. We have reported that VIP inhibited the expressions of TREM-1 and IL-17A, which are involved in the initiation and amplification of inflammation in acute lung injury (ALI). However, the overall effect of VIP on ALI remains unknown. The aim of this study is to investigate the therapeutic effect of VIP mediated by lentivirus (Lenti-VIP) on lipopolysaccharide (LPS)-induced murine ALI. We found that the expression of intrapulmonary VIP peaked at day7 after the intratracheal injection of Lenti-VIP. Lenti-VIP increased the respiratory rate, lung compliance, and tidal volume, while decreased airway resistance in ALI mice, detected by Buxco system. Lenti-VIP significantly reduced inflammatory cell infiltration and maintained the integrity of the alveolar septa. Lenti-VIP also remarkably decreased the total protein level, the number of neutrophil and lactate dehydrogenase activity in the bronchoalveolar lavage fluid of LPS-induced ALI mice. In addition, Lenti-VIP down-regulated pro-inflammatory tumor necrosis factor (TNF)-α mRNA and protein expression, while up-regulated anti-inflammatory interleukin-10 mRNA and protein expression in lungs of ALI mice. Furthermore, we observed that VIP reduced the TNF-α expression in murine macrophages under LPS stimulation through protein kinase C and protein kinase A pathways. Together, our findings show that in vivo administration of lentivirus expressing VIP exerts a potent therapeutic effect on LPS-induced ALI in mice via inhibiting inflammation.

Trefoil Factor 2 Promotes Type 2 Immunity and Lung Repair through Intrinsic Roles in Hematopoietic and Nonhematopoietic Cells

Trefoil factors (TFFs) are small secreted proteins that regulate tissue integrity and repair at mucosal surfaces, particularly in the gastrointestinal tract. However, their relative contribution(s) to controlling baseline lung function or the extent of infection-induced lung injury are unknown issues. With the use of irradiation bone marrow chimeras, we found that TFF2 produced from both hematopoietic- and nonhematopoietic-derived cells is essential for host protection, proliferation of alveolar type 2 cells, and restoration of pulmonary gas exchange after infection with the hookworm parasite Nippostrongylus brasiliensis. In the absence of TFF2, lung epithelia were unable to proliferate and expressed reduced lung mRNA transcript levels for type 2 response-inducing IL-25 and IL-33 after infectious injury. Strikingly, even in the absence of infection or irradiation, TFF2 deficiency compromised lung structure and function, as characterized by distended alveoli and reduced blood oxygen levels relative to wild-type control mice. Taken together, we show a previously unappreciated role for TFF2, produced by either hematopoietic or nonhematopoietic sources, as a pro-proliferative factor for lung epithelial cells under steady-state and infectious injury conditions.

The effect of a single anti-Vascular Endothelial Growth Factor injection on neonatal growth and organ development: In-vivo study

Retinopathy of prematurity (ROP) is one of the leading causes of blindness in preterm Infants. Anti-vascular endothelial growth factor (VEGF) is emerging as a promising treatment, but there is insufficient evidence on their safety. We investigate the effect of systemic anti-VEGF in rat pups with equivalent maturity to a 32 week neonate. A single dose of either anti-VEGF antibody (n = 7) or saline (control group; n = 6) was administered to newborn rats intra-peritoneally on the first day of life. 14 days' post treatment, the serum concentration of anti-VEGF was measured and the brain, lung, heart, kidney and liver were harvested and weighed. The heart was processed to measure the Fulton index (a surrogate for pulmonary hypertension). All other organs were processed for mRNA expression of VEGF and VEGF-receptors (R1&R2). No group differences in body and organ weights were noted. The anti-VEGF was still detected in serum 14 days post Injection and resulted in increase in lung (p < 0.002) and kidney (p < 0.01) VEGF mRNA expressions and the lung (p < 0.02) VEGF-R1 and kidney (P < 0.001) VEGF-R2 mRNA expressions. The treated pups exhibited increased total heart weight (p < 0.01) and Fulton Index (p < 0.05). No changes were seen in the liver and brain. Anti-VEGF antibody did not affect mortality, total body and organ weights, but was associated with pulmonary hypertension. Expression of lung and kidney VEGF and its receptors was increased, whilst the brain and liver did not show changes. Dosing experiments can now be targeted to assess safety threshold and at anti-VEGF dose used in human ROP treatment.

Celastrol Alleviates Airway Hyperresponsiveness and Inhibits Th17 Responses in Obese Asthmatic Mice.

Severe airway hyperresponsiveness (AHR) is a clinical feature of asthma, which has been associated with obesity and has shown a poor response to standard asthma treatments such as glucocorticoids. Numerous studies have shown that Interleukin (IL)-17 producing CD4+T cells (Th17 cells), which could be inhibited by celastrol, is essential in mediating steroid-resistant AHR. The following study investigates the impact of celastrol and its mechanism on the regulation of AHR in murine model of obesity and asthma. C57BL/6 mice were sensitized by intraperitoneal injection of ovalbumin (OVA) on day 1 and 13 starting from 12th week, which was followed by aerosol OVA challenge that lasted for 30 min per daily for 7 consecutive days starting from 16th week. Diet-induced obesity (DIO) mice were fed a high fat diet (HFD) for 16 weeks. Celastrol was administrated orally for 7 consecutive days, 30 min before every challenge in DIO-OVA-induced mice. Lung functions were analyzed by measuring the airway resistance (Rn) and methacholine (MCh) AHR, while H&E staining was used to examine histological changes in the lungs. Immunohistochemistry was used to observe IL-17A protein in lung tissues; flow cytometry to detect the proportion of Th17 cells in CD4+T cells. The concentration of cytokines IL-17A in serum was assessed by standardized sandwich ELISA, while the expression of IL-17A mRNA in lung was examined by quantitative real-time RT-PCR. Briefly, our data indicated that celastrol reduced body mass in DIO-OVA-induced obesity and asthma. Both baseline Rn and MCh AHR were significantly lower in celastrol group. Moreover, celastrol treatment decreased the frequency of Th17 cell expansion and reduced the production of IL-17A in both lung and serum. To sum up, our findings indicated that Th17 and its cytokine measured in the spleen and lung were closely associated with AHR. In addition, celastrol has shown the ability to suppress AHR through Th17 inhibition in obese asthmatic mice.

Regulation of influenza virus replication by Wnt/β-catenin signaling.

Wnt/β-catenin signaling is an essential pathway in cell cycle control. Dysregulation of the Wnt/β-catenin signaling pathway during viral infection has been reported. In this study, we examined the effect of modulating Wnt/β-catenin signaling during influenza virus infection. The activation of the Wnt/β-catenin pathway by Wnt3a increased influenza virus mRNA and virus production in in vitro in mouse lung epithelial E10 cells and mRNA expresson of influenza virus genes in vivo in the lungs of mice infected with influenza virus A/Puerto Rico/8/34. However, the inhibition of Wnt/β-catenin signaling by iCRT14 reduced virus titer and viral gene expression in human lung epithelial A549 cells and viral replication in primary mouse alveolar epithelial cells infected with different influenza virus strains. Knockdown of β-catenin also reduced viral protein expression and virus production. iCRT14 acts at the early stage of virus replication. Treatment with iCRT14 inhibited the expression of the viral genes (vRNA, cRNA and mRNA) evaluated in this study. The intraperitoneal administration of iCRT14 reduced viral load, improved clinical signs, and partially protected mice from influenza virus infection.

Antiasthmatic Effects of Sanglong Pingchuan Decoction through Inducing a Balanced Th1/Th2 Immune Response.

Objective To investigate the antiasthmatic effects of Sanglong pingchuan decoction (SLPCD) and to explore its mechanisms of action. Methods The serum, bronchoalveolar lavage fluid (BALF), and lung tissues from OVA-induced allergic asthma mice were collected 24 h after the last administration. Lung pathological changes were observed by H&E staining. The inflammatory cells in BALF were counted by flow cytometry. The levels of total IgE in serum and cytokines in BALF were determined by ELISA. The expression levels of cytokine mRNA in lung were assayed by qRT-PCR. Results SLPCD significantly inhibited airway inflammation, reduced inflammatory cells in BALF, reduced the levels of total IgE in serum and Th2 cytokines (IL-10 and IL-13) in BALF, and downregulated the mRNA expression levels of Th2 cytokines (IL-4, IL-5, IL-10, and IL-13) in lung of asthmatic mice. However, SLPCD remarkably elevated the level of Th1 cytokine IFN-γ in BALF and upregulated the mRNA expression levels of Th1 cytokines (IL-2 and IFN-γ) in lung of asthmatic mice. Conclusion SLPCD could attenuate airway inflammation and alleviate the pathogenesis in asthma mice through inducing a balanced Th1/Th2 response and could act as an effective drug for treatment of asthma.

Adaptive immune responses are altered in adult mice following neonatal hyperoxia.

Premature infants with bronchopulmonary dysplasia (BPD), are at risk for frequent respiratory infections and reduced pulmonary function. We studied whether neonatal hyperoxia disrupts adaptive immune responses in adult mice, contributing to higher respiratory‐related morbidities seen in these infants. Newborn mice litters were randomized at 3 days to 85% O2 or room air (RA) for 12 days. Whole lung mRNA was isolated in both the groups at 2 weeks and 3 months. Gene expression for T‐cell and B‐cell adaptive immune response was performed by real‐time PCR and qRT‐PCR; protein expression (p21, IL4, IL10, IL27, cd4) was performed by enzyme immunoassay along with p21 immunohistochemistry. Hyperoxia increased expression of p21 and decreased expression of 19 genes representing T/B‐cell activation by ≥ fourfold; three of them significantly (Rag1, Cd1d1, Cd28) compared to the RA group at 2 weeks. Despite RA recovery, the expression of IFN γ, IL27, and CD40 was significantly reduced at 3 months in the hyperoxia group. Expression of p21 was significantly higher and IL27 protein lower at 2 weeks following hyperoxia. Adult mice exposed to neonatal hyperoxia had lower IL4 and IL10 in the lung at 3 months. Adaptive immune responses are developmentally regulated and neonatal hyperoxia suppresses expression of genes involved in T‐/B‐cell activation with continued alterations in gene expression at 3 months. Dysfunction of adaptive immune responses increases the risk for susceptibility to infection in premature infants.

Enhanced Expression of Robo4 Ameliorates LPS Induced Acute Lung Injury in Mice through Binding to Rhoa

Robo4 maintains the barrier function of the mature vascular network by inhibiting endothelial permeability induced by pro-angiogenic factor and the function of Robo4 was tissue specific. The aim of this study was to investigate the role and signaling pathways of Robo4 in pulmonary endothelial permeability in Lipopolysaccharides (LPS) induced acute lung injury (ALI) in mice. Mice were challenged by intra-peritoneal (IP) injection of LPS (15 mg/kg) 48 h after gene transfer of Retro-hRobo4. Protein in bronchoalveolar lavage fluid (BALF), lung wet/dry ratio, lung injury score, MPO activity, secondary cytokine and survival rate was recorded. Co-immunoprecipitation (Co-ip) was used to detect the binding of Robo4 and RhoA. Robo4 mRNA and protein concentration in mice lungs were significantly increased after retrovirus-mediated gene delivery. Up-regulation of Robo4 significantly decreased protein in BALF, lung wet/dry ratio, lung injury score and MPO activity while intercellular tight junction protein ZO-1 was significantly increased. However, up-regulation of Robo4 didn’t change the concentration of TNF-a, vWF and VEGF in serum of mice. In addition, administration of Retro-hRobo4 significantly improved the survival of mice. Robo4 was bind to RhoA confirmed by Co-ip. These findings suggest that up-regulation of Robo4 enhanced pulmonary micro-vascular integrity and reduced lung edema in LPS induced ALI partially through binding to RhoA.

Mesenchymal Stromal Cell Exosomes Ameliorate Experimental Bronchopulmonary Dysplasia and Restore Lung Function through Macrophage Immunomodulation.

Mesenchymal stem/stromal cell (MSC) therapies have shown promise in preclinical models of pathologies relevant to newborn medicine, such as bronchopulmonary dysplasia (BPD). We have reported that the therapeutic capacity of MSCs is comprised in their secretome, and demonstrated that the therapeutic vectors are exosomes produced by MSCs (MSC-exos). To assess efficacy of MSC-exo treatment in a preclinical model of BPD and to investigate mechanisms underlying MSC-exo therapeutic action. Exosomes were isolated from media conditioned by human MSC cultures. Newborn mice were exposed to hyperoxia (HYRX; 75% O2), treated with exosomes on Postnatal Day (PN) 4 and returned to room air on PN7. Treated animals and appropriate controls were harvested on PN7, -14, or -42 for assessment of pulmonary parameters. HYRX-exposed mice presented with pronounced alveolar simplification, fibrosis, and pulmonary vascular remodeling, which was effectively ameliorated by MSC-exo treatment. Pulmonary function tests and assessment of pulmonary hypertension showed functional improvements after MSC-exo treatment. Lung mRNA sequencing demonstrated that MSC-exo treatment induced pleiotropic effects on gene expression associated with HYRX-induced inflammation and immune responses. MSC-exos modulate the macrophage phenotype fulcrum, suppressing the proinflammatory "M1" state and augmenting an antiinflammatory "M2-like" state, both in vitro and in vivo. MSC-exo treatment blunts HYRX-associated inflammation and alters the hyperoxic lung transcriptome. This results in alleviation of HYRX-induced BPD, improvement of lung function, decrease in fibrosis and pulmonary vascular remodeling, and amelioration of pulmonary hypertension. The MSC-exo mechanism of action is associated with modulation of lung macrophage phenotype.

Inhibition of the Receptor for Advanced Glycation End-Products in Acute Respiratory Distress Syndrome: A Randomised Laboratory Trial in Piglets

Background: The receptor for advanced glycation end products (RAGE) modulates the pathogenesis of acute respiratory distress syndrome (ARDS). RAGE inhibition was recently associated with attenuated lung injury and restored alveolar fluid clearance (AFC) in a mouse model of ARDS. However, clinical translation will first require assessment of this strategy in larger animals. Methods: Forty-eight anaesthetised Landrace piglets were randomised into a control group and three treatment groups. Animals allocated to treatment groups underwent orotracheal instillation of hydrochloric acid i) alone; ii) in combination with intravenous administration of a RAGE antagonist peptide (RAP), a S100P-derived peptide that prevents activation of RAGE by its ligands, or iii) in combination with intravenous administration of recombinant soluble (s)RAGE that acted as a decoy receptor. The primary outcome measure was net AFC at 4 h. Arterial oxygenation was assessed hourly for 4 h and alveolar-capillary permeability, alveolar inflammation, lung histology and lung mRNA expression of the epithelial sodium channel (α1-ENaC), α1-Na,K-ATPase and aquaporin (AQP)-5 were assessed at 4 h. Findings: Treatment with either RAP or sRAGE improved net AFC rates (median [interquartile range], 21.2 [18.8-21.7] and 19.5 [17.1-21.5] %/h, respectively, versus 12.6 [3.2-18.8] %/h in injured, untreated controls), improved oxygenation and decreased alveolar inflammation and histological evidence of tissue injury after acid-induced ARDS. RAGE inhibition also restored lung mRNA expression of α1-Na, K-ATPase and AQP-5. Interpretation: RAGE inhibition restored AFC and attenuated lung injury in a piglet model of acid-induced ARDS. Funding: Auvergne Regional Council, Agence Nationale de la Recherche, Direction Generale de l'Offre de Soins. Declaration of Interest: No conflict of interest, other source of financial support, corporate involvement, patent holdings, etc. is to be declared for all authors. Ethical Approval: Animals were maintained and all procedures were performed with the approval of the ethics committee of the French Ministere de l’Education Nationale, de l’Enseignement Superieur et de la Recherche in the Centre International de Chirurgie Endoscopique, School of Medicine - University of Clermont-Ferrand (approval number 01505.03). All experiments were performed in accordance with the “Animal Research: Reporting In Vivo Experiments” (ARRIVE) guidelines.

Protective effect of hydroxysafflor yellow A on bleomycin- induced pulmonary inflammation and fibrosis in rats.

To observe the effect of hydroxysafflor yellow A (HSYA), an active ingredient of a traditional Chinese herbal medicine Carthamus tinctorius L., on lung inflflammation and pulmonary fibrosis induced by bleomycin (BLM) in rats. Animals were divided into 6 groups including normal group, model group, three HSYA groups and dexamethasone (DXM) group. Three doses of HSYA (35.6, 53.3, and 80.0 mg•kg-1•day-1) were intraperitoneally (i.p.) injected in rats for 3 weeks after BLM administration and DXM was used as the positive control (n=8 or 12). Arterial blood gas was assayed and morphological changes were observed. Lung mRNA expressions of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and some cytokines in lung tissue were detected by real-time polymerase chain reaction. Nuclear factor-κB p65 or α-smooth muscle actin (α-SMA) protein distribution in rat lung tissue was observed by immunohistochemistry. On the 7th day after BLM administration, lung tissue showed serious inflammation. Treatment with HSYA or DXM ameliorated lung inflammation. After treatment with HSYA or DXM, oxygen partial pressure (PaO2) increased (HSYA 80.0 mg•kg-1, P<0.01) and CO2 partial pressure (PaCO2) decreased (HSYA 53.3, 80.0 mg•kg-1, P<0.05). Moreover, the mRNA expression of TNF-α, IL-1β, and IL-6; and the number of NF-κB p65 positive cells was lower in HSYA 53.3 and 80.0 mg•kg-1 groups than those in the model group (all P<0.05). Twenty-one days after BLM administration, HSYA or DXM treatment ameliorated fibrosis, increased PaO2 (HSYA 53.3, 80.0 mg•kg-1, P<0.01), and decreased PaCO2 (53.3 and 80.0 mg•kg-1, P<0.05). Further, the mRNA expression of TGF-β1, α-SMA, and collagen I as well as the number of α-SMA positive cells increased in the model group and HSYA can attenuate these changes (53.3, 80.0 mg•kg-1, P<0.05). Hematoxylin and eosin and Masson's trichrome staining indicated that the fibrosis and collagen deposition were ameliorated in HSYA groups (53.3, 80.0 mg•kg-1, P<0.05). HSYA could alleviate acute lung inflflammation and chronic pulmonary fibrosis induced by BLM in rats.

The SOD3 R213G Polymorphism Promotes Resolution of Innate Immune Activation and Blocks Induction of miR29b in Bleomycin Induced Lung Inflammation and Fibrosis

A naturally occurring single nucleotide polymorphism (SNP) (R213G) in extracellular superoxide dismutase (SOD3), a key lung and vascular antioxidant enzyme, decreases tissue binding and increases SOD3 in epithelial lining fluid and plasma. Mice harboring the R213G SNP are protected against bleomycin-induced alveolar inflammation and fibrosis. We hypothesized that the redistribution of SOD3 due to the R213G SNP, through site specific changes in the redox environment, modulates key miRNA and mRNA signaling networks responsible for macrophage activation and fibrosis. Plasma, lung and BALF O2•‒ was measured by electron paramagnetic resonance using the CMH spin probe. Bleomycin increased O2•‒ in all three compartments; the increase in O2•‒ was attenuated in the blood of R213G mice. We performed high throughput RNAseq and bioinformatics analyses in lung mRNA and miRNA 7 days post-bleomycin in WT and R213G mice. We identified distinct mRNA and miRNA profiles between strains in response to bleomycin. By pathway analysis, key immune and inflammatory pathways were activated in WT mice but suppressed in R213G mice at 7 days, the timepoint associated with resolution of inflammation in the R213G mice. Predicted upstream regulators included multiple signaling molecules within the TLR4/NF-ks/IKKe/IRF3 pathway. Distinct strain specific miRNA responses were observed, including miR29, a key miRNA implicated in fibrosis, which decreased only in R213G mice. In bone marrow derived macrophages (BMDM) from WT and R213G mice, the response to classic “M1” LPS + IFN polarizing stimuli was similar between strains but attenuated by pretreatment with exogenous extracellular SOD. Using bioinformatics and molecular approaches, we conclude that the redistribution of ecSOD, due to its impact on O2•‒ , can suppress innate immune responses by regulating macrophage plasticity, accounting for the ability of the R213G SNP to protect against bleomycin-induced lung fibrosis. The role of miRNA regulation in this process requires further study.

Nrf2 transfection enhances the efficacy of human amniotic mesenchymal stem cells to repair lung injury induced by lipopolysaccharide.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are clinical emergencies with no effective pharmaceutical treatment. This study aims to determine the protective effects of Nrf2-transfected human amniotic mesenchymal stem cells (hAMSCs) against lipopolysaccharide (LPS)-induced lung injury in mice. hAMSCs stably transfected with Nrf2 or green fluorescent protein control were transplanted into male C57BL/6 mice via the tail vein 4 h after intratracheal instillation of LPS. At 3, 7, and 14 days after cell transplantation, total lung injury score (the Smith score) was determined by hematoxylin and eosin staining. Lung fibrosis was assessed by Masson's trichrome staining. Alveolar epithelial apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick end labeling staining. The plasma levels of interleukin (IL)-1β, IL-6, and IL-10 were determined by enzyme-linked immunosorbent assays (ELISA). The homing and differentiation of hAMSCs into type II alveolar epithelial (AT II) cells were examined by immunofluorescent staining and/or western blot analysis. Nrf2, mRNA, and protein expression in lungs were examined by qRT-PCR and western blot analysis, and DNA-binding activity of Nrf2 was detected by ELISA. We found that, compared with control hAMSCs, treatment with Nrf2-overexpressing hAMSCs led to further reduced lung injury, lung fibrosis, and inflammation in LPS-challenged mice. Nrf2-overexpressing hAMSCs also exhibited increased cell retention in the lung, more efficient differentiation into AT II cells, and more prominent effects on the increased mRNA and protein expression as well as DNA-binding activity of Nrf2 than control. These results support Nrf2-overexpressing hAMSCs as a potential cell-based therapy for clinical ALI/ARDS.

Targeted silencing of SOX2 by an artificial transcription factor showed antitumor effect in lung and esophageal squamous cell carcinoma.

SOX2 is a transcription factor essential for early mammalian development and for the maintenance of stem cells. Recently, SOX2 was identified as a lineage specific oncogene, recurrently amplified and activated in lung and esophageal squamous cell carcinoma (SCC). In this study, we have developed a zinc finger-based artificial transcription factor (ATF) to selectively suppress SOX2 expression in cancer cells and termed the system ATF/SOX2. We engineered the ATF using six zinc finger arrays designed to target a 19 bp site in the SOX2 distal promoter and a KOX transcriptional repressor domain. A recombinant adenoviral vector Ad-ATF/SOX2 that expresses ATF/SOX2 suppressed SOX2 at the mRNA and protein levels in lung and esophageal SCC cells expressing SOX2. In these kinds of cells, Ad-ATF/SOX2 decreased cell proliferation and colony formation more effectively than the recombinant adenoviral vector Ad-shSOX2, which expresses SOX2 short hairpin RNA (shSOX2). Ad-ATF/SOX2 induced the cell cycle inhibitor CDKN1A more strongly than Ad-shSOX2. Importantly, the ATF did not suppress the cell viability of normal human cells. Moreover, Ad-ATF/SOX2 effectively inhibited tumor growth in a lung SCC xenograft mouse model. These results indicate that ATF/SOX2 would lead to the development of an effective molecular-targeted therapy for lung and esophageal SCC.

Attenuation of hemorrhage-associated lung injury by adjuvant treatment with C23, an oligopeptide derived from cold-inducible RNA-binding protein.

Hemorrhagic shock (HS) is an important cause of mortality. HS is associated with an elevated incidence of acute lung injury and acute respiratory distress syndrome, significantly contributing to HS morbidity and mortality. Cold-inducible RNA-binding protein (CIRP) is released into the circulation during HS and can cause lung injury. C23 is a CIRP-derived oligopeptide that binds with high affinity to the CIRP receptor and inhibits CIRP-induced phagocyte secretion of TNF-α. This study was designed to determine whether C23 is able to attenuate HS-associated lung injury. C57BL/6 mice were subjected to controlled hemorrhage leading to a mean arterial pressure of 25 ± 3 mm Hg for 90 minutes. Mice were then volume-resuscitated for 30 minutes with normal saline solution alone (vehicle) or plus adjuvant treatment with C23 (8 mg/kg BW). At 4.5 hours after resuscitation, the blood and lungs were harvested. Serum levels of organ injury markers lactate dehydrogenase, aspartate aminotransferase were significantly elevated in hemorrhaged mice receiving vehicle and were reduced by 51.3% and 52.2% in mice adjuvantly treated with C23, respectively. Similarly, lung mRNA levels of IL-1β, TNF-α, and IL-6, and lung myeloperoxidase activity were elevated after HS and reduced by 66.1%, 54.4%, 69.7%, and 24.3%, respectively, in mice treated with C23. Adjuvant treatment with C23 also decreased the lung histology score by 33.9%, lung extravasation of albumin carrying Evans blue dye by 36.8%, and the protein level of intercellular adhesion molecule-1, and indicator of vascular endothelial cell activation, by 40.3%. Together, these results indicate that adjuvant treatment with the CIRP-derived oligopeptide C23 is able to improve lung inflammation and vascular endothelial activation secondary to HS, lending support to the development of CIRP-targeting adjuvant treatments to minimize lung injury after HS.

CISH controls bacterial burden early after infection with Mycobacterium tuberculosis in mice

CISH gene has been associated with increased susceptibility to human tuberculosis. We found that cish−/− mice had higher M. tuberculosis load in spleens and lungs up to 2.5 weeks after infection but not later compared to controls. Cish mRNA levels were increased in lungs at early and late time points after M. tuberculosis infection. In relation, the titers of inos and tnf mRNA in lungs were reduced early after infection of cish−/− mice.The transfer of cish−/− and control T cells conferred rag1−/− mice similar protection to infection with M. tuberculosis.Macrophages showed increased cish mRNA levels after M. tuberculosis infection in vitro. However, mycobacterial uptake and growth in cish−/− and control macrophages was similar.Thus, we here show that CISH mediates control of M. tuberculosis in mice early after infection via regulation of innate immune mechanisms.

Abstract P248: Novel Role of Axl Kinase in Endothelial Cell Proliferation and Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a poorly characterized disease of unclear etiology that affects individuals of all ages. Vascular remodeling and increase in pulmonary artery (PA) and right ventricle (RV) pressures are two major culprits in RV failure and death in PAH. Recent advances in the study of PAH suggest that endothelial cell proliferation is an early instigator of this hallmark remodeling. We postulated that Axl receptor tyrosine kinase (implicated in pro-proliferative and pro-survival signaling in cancerous cells) could mediate endothelial proliferation and thus hemodynamic changes occurring in PAH. Using immunofluorescent microscopy of lung microvessels of human PAH vs. non-PAH, we observed Axl expression on intimal endothelial cells but not medial smooth muscle cells. Furthermore, digitized microscopy revealed that Axl tended to increase on the endothelium of PAH vessels (1.65±0.15-fold vs. non-PAH; n=3-4; p=0.057 ). To address the role of Axl in vivo , an Axl inhibitor R428 was employed in a mouse model of pulmonary hypertension. C57Bl/6 mice were subjected to hypoxia at pO 2 =10% and VEGF receptor antagonist SU5416 (Su/Ch) or normoxia (Norm) for 3 wks. Indeed, Su/Ch caused a significant rise in lung Axl protein and mRNA (7.1±0.4- and 2.4±0.5-fold, Su/Ch vs. Norm, protein and mRNA, respectively; n=3-6; p&lt;0.01). As predicted, RV pressure (RVP) rose from 27±0.5 to 43±1.8 mmHg (Norm vs. Su/Ch; n=6; p&lt;0.01). However, we did not observe a decrease in RVP with twice-daily gavage of 75 mg/kg R428 (43±1.4 mmHg, Su/Ch + R428; n=6). A similar pattern was observed with mean PA pressure (18.4±0.3 and 28.7±1.2 mmHg, Norm vs. Su/Ch, p&lt;0.01; 28.7±0.9 mmHg, Su/Ch + R428), RV resistance (1403±256 vs. 2703±464 Wood units, Norm vs. Su/Ch, n/s; vs. 3610±625 Wood units, Su/Ch + R428) and Fulton index (0.26±0.01 and 0.34±0.02, Norm vs. Su/Ch, p&lt;0.05; 0.38±0.02, Su/Ch + R428). In conclusion, our preliminary results demonstrate upregulated Axl expression in the endothelium of PAH patients and in lungs of PH mice and suggest that Axl kinase may play a novel role in pulmonary vascular endothelial proliferation and remodeling in PAH. It remains to be determined whether drug bioavailability or severity of disease precluded an ameliorative effect of an Axl inhibitor.

Vitamin D supplementation of initially vitamin D-deficient mice diminishes lung inflammation with limited effects on pulmonary epithelial integrity.

In disease settings, vitamin D may be important for maintaining optimal lung epithelial integrity and suppressing inflammation, but less is known of its effects prior to disease onset. Female BALB/c dams were fed a vitamin D3‐supplemented (2280 IU/kg, VitD+) or nonsupplemented (0 IU/kg, VitD−) diet from 3 weeks of age, and mated at 8 weeks of age. Male offspring were fed the same diet as their mother. Some offspring initially fed the VitD− diet were switched to a VitD+ diet from 8 weeks of age (VitD−/+). At 12 weeks of age, signs of low‐level inflammation were observed in the bronchoalveolar lavage fluid (BALF) of VitD− mice (more macrophages and neutrophils), which were suppressed by subsequent supplementation with vitamin D3. There was no difference in the level of expression of the tight junction proteins occludin or claudin‐1 in lung epithelial cells of VitD+ mice compared to VitD− mice; however, claudin‐1 levels were reduced when initially vitamin D‐deficient mice were fed the vitamin D3‐containing diet (VitD−/+). Reduced total IgM levels were detected in BALF and serum of VitD−/+ mice compared to VitD+ mice. Lung mRNA levels of the vitamin D receptor (VDR) were greatest in VitD−/+ mice. Total IgG levels in BALF were greater in mice fed the vitamin D3‐containing diet, which may be explained by increased activation of B cells in airway‐draining lymph nodes. These findings suggest that supplementation of initially vitamin D‐deficient mice with vitamin D3 suppresses signs of lung inflammation but has limited effects on the epithelial integrity of the lungs.

Marmoset pulmonary cytochrome P450 2F1 oxidizes biphenyl and 7-ethoxycoumarin and hepatic human P450 substrates

1. A potentially useful animal model for preclinical studies is the common marmoset (Callithrix jacchus). In this study, using reverse-transcription polymerase chain reaction from marmoset livers, we identified a novel cytochrome P450 (P450) 2F1 cDNA with an open reading frame of 1473 bp.2. High sequence identities of 92-94% with primate P450 2 F amino acid sequences were indicated by deduced amino acid sequences of P450 2F1 cDNA. Phylogenetic analysis indicates that marmoset P450 2F1 is more congruent with primate P450 2 F forms than those of other species such as rodents.3. Among five tissue types examined, abundant expression of marmoset P450 2F1 mRNA and P450 2F1 protein in lungs was shown. Cynomolgus monkey P450 2F1 mRNA was abundantly expressed in lungs as well as testes and ovaries in 10 tissue types.4. Similar to those of humans and cynomolgus monkeys, marmoset P450 2F1 heterologously expressed in Escherichia coli membranes efficiently catalyzed 7-ethoxycoumarin O-deethylation and biphenyl hydroxylation, however unlike human P450 2F1, marmoset P450 2F1 exhibited hydroxylation activity toward coumarin and chlorzoxazone.5. These findings indicated that P450 2F1 enzyme expressed in marmoset lungs and also catalyzed metabolism of xenobiotics, suggesting the importance of P450 2 F-dependent drug metabolism in marmoset lungs.

Significance of the NLRP3 inflammasome expression in rats with acute lung injury induced by phosgene

Objective: To investigate changes of NLRP3 signal transduction pathway of acute lung injury induced by phosgene to analyze NLRP3-mediated IL-1β release inflammatory process in rats. Methods: Rats were randomly divided into two groups, 10 rats in the Air group that consists of the rats with air exposure, 10 rats in the Psg group that consists of the rats with phosgene exposure at 8.33 g/m(3) for 5 min. The specimens of serum, bronchoalveolar lavage fluid (BALF) and lung were collected after 6h. Morphological changes were observed by HE staining. The expression of NLRP3 in the lung of two groups was detected by immunohistochemistry. NLRP3、ASC and caspase-1 expression in the lung tissue was quantified by Western blot. Reverse transcription-polymerase chain reaction (RT-PCR) were used to detect the expression of NLRP3、ASC and caspase-1 mRNA in the lung tissue. The concentrations of IL-1β、IL-18 and IL-33 in the serum and BALF were measured by enzyme-linked immunosorbent assay. RT-PCR were used to detect the expression of IL-1β、IL-18 and IL-33 mRNA in the lung tissue. Results: We successfully replicated the model of phosgene-induced ALI in rats. Morphological of HE staining after phosgene exposure to 6 h observed inflammatory cell infiltration in lung tissue in Phosgene group. Immunohistochemical staining results showed that there were many NLRP3 positive cells in lung tissue in Phosgene group. The levels of NLRP3 and caspase-1 mRNA and protein expression in lung were significantly increased (P<0.05) in Phosgene group, but no significant change was observed in lung ASC mRNA and protein expression (P>0.05) . Compared with Air group, the serum, BALF and lung tissue of IL-1β、IL-18 and IL-33 mRNA and protein expression were significantly increased (P<0.01) in Phosgene group. Conclusion: NLRP3-mediated inflammatory response probably involved in the process of the phosgene, so it maybe one of the pathogenesis of acute lung injury.