Intratracheal Lipopolysaccharide Research Articles

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

Astragalin reduces lipopolysaccharide-induced acute lung injury in rats via induction of heme oxygenase-1.

Astragalin, a bioactive component of medicinal plants such as Rosa agrestis, has anti-inflammatory and antioxidant features. Induction of heme oxygenase (HO)-1 is an effective strategy to reduce excessive generated oxidants during the pathogenesis of acute lung injury (ALI). The aim of the present study is to investigate that whether the anti-inflammatory and antioxidant features of astragalin is HO-1 dependent in lipopolysaccharide (LPS)-induced ALI. Sprague-Dawley rats were used in animal study. Intratracheal LPS was performed to induce experimental ALI model. Astragalin was administrated 1h after LPS challenge. Human lung epithelial cells were used in cell study. Samples from rats were harvested at 24h post LPS challenge. Astragalin treatment inhibited LPS-induced inflammatory cells infiltration in the lung and pulmonary edema. Astragalin treatment markedly enhanced the activity of HO-1 compared with vehicle-treated group at 24h post LPS challenge. Levels of lipid hydroperoxide, a marker for oxidative stress, were decreased in astragalin-treated animals compared with vehicle-treated group. However, the protective effect of astragalin on LPS-induced ALI was abolished in an inhibitor of HO-1-treated animals. Moreover, the astragalin-induced the upregulation of HO-1 in human lung epithelial cells was inhibited when nuclear factor erythroid-2-related factor 2 (Nrf2) was silenced by small interfering RNA. Astragalin reduces LPS-induced ALI via activation of Nrf2/HO-1 pathway.

An inflammatory pulmonary insult post-traumatic brain injury worsens subsequent spatial learning and neurological outcomes.

Severe traumatic brain injury (TBI) patients are at high risk for early aspiration and pneumonia. How pneumonia impacts neurological recovery after TBI is not well characterized. We hypothesized that, independent of the cerebral injury, pneumonia after TBI delays and worsens neurological recovery and cognitive outcomes. Fifteen CD1 male mice were randomized to sham craniotomy or severe TBI (controlled cortical impact [CCI] - velocity 6 m/s, depth 1.0 mm) ± intratracheal lipopolysaccharide (LPS-2 mg/kg in 0.1 mL saline) as a pneumonia bioeffector. Neurological functional recovery by Garcia Neurologic Testing (GNT) and body weight loss were recorded daily for 14 days. On Days 6-14, animals underwent Morris Water Maze learning and memory testing with cued trials (platform visible), spatial learning trials (platform invisible, spatial cues present), and probe (memory) trials (platform removed, spatial clues present). Intergroup differences were assessed by the Kruskal-Wallis test with Bonferroni correction (p < 0.05). Weight loss was greatest in the CCI + LPS group (maximum 24% on Day 3 vs. 8% [Sham], 7% [CCI], both on Day 1). GNT was lowest in CCI + LPS during the first week. Morris Water Maze testing demonstrated greater spatial learning impairment in the CCI + LPS group vs. Sham or CCI counterparts. Cued learning and long-term memory were worse in CCI + LPS and CCI as compared to Sham. A pneumonia bioeffector insult after TBI worsens weight loss and mortality in a rodent model. Not only is spatial learning impaired, but animals are more debilitated and have worse neurologic performance. Understanding the adverse effects of pneumonia on TBI recovery is the first step d patients.

A Peptide Inhibitor of NADPH Oxidase (NOX2) Activation Markedly Decreases Mouse Lung Injury and Mortality Following Administration of Lipopolysaccharide (LPS).

We have previously derived three related peptides, based on a nine-amino acid sequence in human or rat/mouse surfactant protein A, that inhibit the phospholipase A2 activity of peroxiredoxin 6 (Prdx6) and prevent the activation of lung NADPH oxidase (type 2). The present study evaluated the effect of these Prdx6-inhibitory peptides (PIP) in a mouse (C57Bl/6) model of acute lung injury following lipopolysaccharide (LPS) administration. All three peptides (PIP-1, 2 and 3) similarly inhibited the production of reactive O2 species (ROS) in isolated mouse lungs as detected by the oxidation of Amplex red. PIP-2 inhibited both the increased phospholipase A2 activity of Prdx6 and lung reactive oxygen species (ROS) production following treatment of mice with intratracheal LPS (5 µg/g body wt.). Pre-treatment of mice with PIP-2 prevented LPS-mediated lung injury while treatment with PIP-2 at 12 or 16 h after LPS administration led to reversal of lung injury when evaluated 12 or 8 h later, respectively. With a higher dose of LPS (15 µg/g body wt.), mortality was 100% at 48 h in untreated mice but only 28% in mice that were treated at 12–24 h intervals, with PIP-2 beginning at 12 h after LPS administration. Treatment with PIP-2 also markedly decreased mortality after intraperitoneal LPS (15 µg/g body wt.), used as a model of sepsis. This study shows the dramatic effectiveness of a peptide inhibitor of Prdx6 against lung injury and mouse mortality in LPS models. We propose that the PIP nonapeptides may be a useful modality to prevent or to treat human ALI.

Responses of retinal arterioles and ciliary arteries in pigs with acute respiratory distress syndrome (ARDS)

Acute respiratory distress syndrome (ARDS) is a clinical syndrome of acute lung failure in critically sick patients, which severely compromises the function of multiple organs, including the brain. Although, the optic nerve and the retina are a part of the central nervous system, the effects of ARDS on these ocular structures are completely unknown. Thus, the major goal of this study was to test the hypothesis that ARDS affects vascular function in the eye. ARDS was induced in anesthetized pigs by intratracheal injection of lipopolysaccharide (LPS). Sham-treated animals served as controls. Pigs were monitored for 8 h and then sacrificed. Subsequently, retinal arterioles and short posterior ciliary arteries were isolated and cannulated with micropipettes to measure vascular responses by videomicroscopy. Levels of reactive oxygen species (ROS) were quantified in isolated vessels using dihydroethidium (DHE). Messenger RNA expression of hypoxic, inflammatory, prooxidative, and antioxidative genes was assessed by real-time PCR. When group-dependent differences in mRNA expression levels were found for a particular gene, immunostainings were conducted. Strikingly, responses to the endothelium-dependent vasodilator, bradykinin, were markedly impaired in retinal arterioles of LPS-treated pigs, but no differences were seen between ciliary arteries of LPS- and sham-treated animals. ROS levels were increased in retinal arterioles but not in ciliary arteries of LPS-treated pigs. Messenger RNA levels for HIF-1α, VEGF-A and NOX2 were markedly increased in retinal arterioles of LPS-treated pigs, whereas ciliary arteries had only negligible mRNA level changes. Pronounced immunoreactivity for HIF-1α, VEGF-A and NOX2 was seen in the endothelium of retinal arterioles from LPS-treated pigs. Histologically, massive edema was seen especially in the retinal nerve fiber layer of pigs treated with LPS. Our study provides the first evidence that ARDS induced by intratracheal LPS application evokes endothelial dysfunction in porcine retinal arterioles together with retinal edema, indicative of vascular leakage. In contrast, ciliary arteries appear to be resistant to intratracheal LPS application.

A CRTH2 antagonist, CT-133, suppresses NF-κB signalling to relieve lipopolysaccharide-induced acute lung injury

Acute lung injury (ALI) and acute respiratory distress syndrome are life-threatening conditions that still have no definite pharmacotherapy. Hence, we investigate the potential effectiveness and underlying mechanism of CT-133, a newly developed selective antagonist of prostaglandin D2 receptor 2 (DP2) or of chemoattractant receptor homologous molecule expressed on Th2 cells (CRTH2), against lipopolysaccharide (LPS)-induced ALI. CT-133 (10 or 30 mg/kg) or dexamethasone (1 mg/kg, positive control) were intragastrically administered 1 h before and 12 h after intratracheal LPS instillation, and primary neutrophils and macrophages and RAW264.7 macrophages were used to investigate the role of CT-133 in regulation of their functions. LPS induced a significant secretion of PGD2 from primary macrophages, however, CT-133 dose-dependently and markedly decreased the infiltration of neutrophils and macrophages into lungs, reduced the IL-1β, TNF-α, IL-6, and KC levels in broncho-alveolar lavage (BAL) fluids, decreased the wet weight and myeloperoxidase activity of lungs, reduced Evans blue and albumin exudation into lungs, and improved the lung histopathological changes and hypoxemia. Moreover, CT-133 significantly suppressed the primary neutrophil migration toward the PGD2 and robustly inhibited the mRNA and protein expression of IL-1β, TNF-α, IL-6, and KC in primary and RAW264.7 macrophages in response to either LPS- or PGD2 stimulation. Finally, CT-133 significantly blocked the LPS-induced P65 activation in both RAW264.7 macrophages and mouse lungs. Thus, This is the first report that a CRTH2 antagonist, CT-133, is capable of significantly alleviating LPS-induced lung injury by probably down-regulating the NF-κB signalling.

Improved Acute Hypoxic Ventilatory Response (HVR) After Pharmacological Activation of 5‐HT1A Receptors in Two‐Hit Lipopolysaccharide (LPS)‐Treated Urethane‐anesthetized Spontaneously Breathing Adult Male Sprague‐Dawley Rat with Prior Single‐Bout Acute Intermittent Hypoxia Exposure

Accumulating evidence indicates that inflammation and serotonergic (5‐HT) neurotransmission interact thereby affecting a variety of physiological processes. Ongoing studies in our laboratory are examining the effects of peripheral inflammation, and its accompanying neuroinflammation, on the neural control of breathing, including the impact of LPS‐induced neuroinflammation on 5‐HT1A receptor‐mediated inspiratory motor behaviors. Here, we examined the effects of systemic administration of the 5‐HT1A receptor agonist 8‐hydroxy‐2‐(di‐npropylamino) tetralin (8‐OH DPAT) on the (diaphragm) EMG burst amplitude and frequency response to a single bout of acute hypoxia (HVR; 11% O2 for 90s) in spontaneously breathing urethane‐anesthetized adult male Sprague‐Dawley rats following a ‘two‐hit’ lipopolysaccharide (LPS) administration protocol in which systemic LPS (3 mg/kg, ip) was administered ~24 hr prior to an intratracheal LPS (0.5 mg/kg, IT) injection. Similar HVR experiments were performed in control rats that were either untreated or received two‐hit saline injections. All rats used for this study had also undergone an acute intermittent hypoxia (AIH) exposure protocol that was completed at least 90 min prior to administration of 8‐OH DPAT, and they were continuously supplied with 2% CO2 throughout the recording protocol. Following baseline (BL; 40% O2) recording of diaphragm EMG activity, 8‐OH DPAT (0.3 mg/kg, iv) was administered and allowed to exert its effects for 30 min, after which a 30s HVR challenge was performed (11% O2) followed by recovery under BL conditions. Data from these experiments were also compared to our previous observations in two‐hit LPS‐ and vehicle‐treated male rats that had not undergone an AIH exposure protocol or received 8‐OH DPAT (to serve as a control for two‐hit LPS HVR behaviors). In brief, we had reported that two‐hit LPS administration blunts the HVR amplitude response such that it increases by ≤10% above BL levels (versus ≥30% increase in vehicle‐treated rats) and produces an exaggerated frequency HVR response such that it increases by ≥30% above BL levels (versus~15–20% increase in vehicle‐treated rats). In contrast to these observations, in the current study, we found that following 8‐OH DPAT administration, the HVR exhibited similar increases in both burst frequency and amplitude in both control and LPS‐treated rats. In this case, HVR amplitude and frequency increased on average by ~23% and ~25%, respectively, although LPS‐treated rats exhibited variable magnitude HVR responses. Regardless, following 8‐OH DPAT administration, HVR inspiratory motor activity appeared to be indistinguishable between control and LPS‐treated rats. These observations suggest that following pharmacological activation of 5‐HT1A receptors, amplitude and frequency HVR behaviors are improved in two‐hit LPS‐treated rats. The specific mechanisms underlying these differences remain to be identified.Support or Funding InformationNIH NS101737; Thomas Hartman Center for Parkinson's Disease Research at Stony Brook UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Recipient CD103+ Classical Dendritic Cells Enhance Acute Rejection in Response to Airway Inflammation after Mouse Lung Transplantation

Purpose Airway inflammation after lung transplantation (LTX), resulting from infection, pollution, or aspiration, is an important risk factor for chronic lung allograft dysfunction. Classical dendritic cells (cDCs) link innate immunity and adaptive immunity, and exhibit regional and functional specialization in the lung. CD103+ cDCs reside in the airway epithelium. Their role in lung transplantation (LTX) has not been extensively investigated. We hypothesized that CD103+ cDCs augment adaptive immune responses under conditions of lipopolysaccharide (LPS)-induced airway inflammation inamouse minor alloantigen-mismatched orthotopic LTX model (C57BL/10 [B10] →C57BL/6 [B6] and vice versa). Methods BATF3-/- mice (B6 background), lacking CD103+ cDCs, were used. Three comparisons were made: 1) B10 → 6 vs. B10 → ATF3-/-, 2) B10 →B6 vs. B10 → ATF3-/- with 6 doses of intra-tracheal (i.t.) LPS (5μg in 50μlPBS)on serial postoperative days (POD), 3)B6 → 10 and BATF3-/- → 10 with 8 doses of i.t. LPS on serial PODs. The grafts were assessed on POD 28. Results Acute rejection (ISHLT criteria) did not differ significantly between B10 → 6 and B10 → ATF3-/- without LPS, or between B6 → 10 and BATF3-/- → 10 with LPS. In contrast, with LPS exposure, acute rejection scores were lower in B10àBATF3-/- compared to B10 àB6 (Figure A). Surprisingly, lung cell analysis by flow cytometry showed that monocytes and neutrophils were significantly higher in BATF3-/- recipients than wild-type recipients under repeated airway LPS exposure (Figure B). Conclusion Our data show that recipient, but not donor, CD103+ cDCs contribute to the augmentation of acute rejection after LTx in the setting of ongoing airway inflammation. As CD103+ cDCs can promote immune regulation, they may be an important therapeutic target after LTX. Airway inflammation after lung transplantation (LTX), resulting from infection, pollution, or aspiration, is an important risk factor for chronic lung allograft dysfunction. Classical dendritic cells (cDCs) link innate immunity and adaptive immunity, and exhibit regional and functional specialization in the lung. CD103+ cDCs reside in the airway epithelium. Their role in lung transplantation (LTX) has not been extensively investigated. We hypothesized that CD103+ cDCs augment adaptive immune responses under conditions of lipopolysaccharide (LPS)-induced airway inflammation inamouse minor alloantigen-mismatched orthotopic LTX model (C57BL/10 [B10] →C57BL/6 [B6] and vice versa). BATF3-/- mice (B6 background), lacking CD103+ cDCs, were used. Three comparisons were made: 1) B10 → 6 vs. B10 → ATF3-/-, 2) B10 →B6 vs. B10 → ATF3-/- with 6 doses of intra-tracheal (i.t.) LPS (5μg in 50μlPBS)on serial postoperative days (POD), 3)B6 → 10 and BATF3-/- → 10 with 8 doses of i.t. LPS on serial PODs. The grafts were assessed on POD 28. Acute rejection (ISHLT criteria) did not differ significantly between B10 → 6 and B10 → ATF3-/- without LPS, or between B6 → 10 and BATF3-/- → 10 with LPS. In contrast, with LPS exposure, acute rejection scores were lower in B10àBATF3-/- compared to B10 àB6 (Figure A). Surprisingly, lung cell analysis by flow cytometry showed that monocytes and neutrophils were significantly higher in BATF3-/- recipients than wild-type recipients under repeated airway LPS exposure (Figure B). Our data show that recipient, but not donor, CD103+ cDCs contribute to the augmentation of acute rejection after LTx in the setting of ongoing airway inflammation. As CD103+ cDCs can promote immune regulation, they may be an important therapeutic target after LTX.

Small-Animal PET/CT Imaging of Local and Systemic Immune Response Using 64Cu-αCD11b.

Current noninvasive imaging methods for monitoring immune response were largely developed for interrogation of the local reaction. This study developed the radiotracer 64Cu-labeled anti-CD11b (64Cu-αCD11b) for longitudinal assessment of local and systemic immune response involving mobilization of CD11b+ myeloid cells by small-animal PET/CT. Methods: Acute or chronic inflammation in the ears of BALB/c mice was induced by 12-o-tetradecanoylphorbol-13-acetate. Acute lung inflammation was induced by intratracheal lipopolysaccharide inoculation. αCD11b was conjugated with p-SCN-Bn-DOTA followed by labeling with 64Cu. PET/CT and biodistribution were evaluated at different times after intravenous injection of 64Cu-αCD11b. Cell populations from bone marrow (BM) and spleen were analyzed by flow cytometry. Results:64Cu-αCD11b was primarily taken up by BM and spleen in control mice. In comparison, 64Cu-αCD11b uptake was significantly reduced in the BM and spleen of CD11b-knockout mice, indicating that 64Cu-αCD11b selectively homed to CD11b+ myeloid cells in vivo. In mice with ear inflammation, for the local inflammatory response, 64Cu-αCD11b PET/CT revealed significantly higher 64Cu-αCD11b uptake in the inflamed ears in the acute inflammation phase than the chronic phase, consistent with markedly increased infiltration of CD11b+ cells into the inflammatory lesions at the acute phase. Moreover, imaging of 64Cu-αCD11b also showed the difference in mouse systemic response for different inflammatory stages. Compared with uptake in control mice, BM 64Cu-αCD11b uptake in mice with ear inflammation was significantly lower in the acute phase and higher in the chronic phase, reflecting an initial mobilization of CD11b+ cells from the BM to the inflammatory foci followed by a compensatory regeneration of CD11b+ myeloid cells in the BM. Similarly, in mice with lung inflammation, 64Cu-αCD11b PET/CT readily detected acute lung inflammation and recruitment of CD11b+ myeloid cells from the BM. Immunohistochemistry staining and flow cytometry results confirmed the noninvasive imaging of PET/CT. Conclusion:64Cu-αCD11b PET/CT successfully tracked ear and pulmonary inflammation in mice and differentiated acute from chronic inflammation at the local and systemic levels. 64Cu-αCD11b PET/CT is a robust quantitative method for imaging of local and systemic immune responses.

Knockdown of TFPI-Anchored Endothelial Cells Exacerbates Lipopolysaccharide-Induced Acute Lung Injury Via NF-κB Signaling Pathway.

ABSTRACTAs activation of the coagulation system is both a consequence and contributor to acute lung injury (ALI), pulmonary coagulopathy has become a potential target for therapeutic intervention in ALI patients. We investigated the effects and possible mechanisms of endothelial cell (EC)-anchored tissue factor pathway inhibitor (TFPI) on lipopolysaccharide (LPS)-induced ALI in mice. To assess the effect of EC-anchored TFPI deletion on ALI indices, TFPI knockout (cKO) mice were generated. Mice were instilled by direct intratracheal injection LPS for the preparation of an ALI model. Evans blue dye (EBD) was injected intravenously 2 h prior to animal sacrifice (48 h post-LPS). Lungs were fixed for histopathology and the prepared tissue was homogenized or used to extract bronchoalveolar lavage fluid (BALF) or detect EBD concentration. TFPI knockdown mice with ALI were compared to wild-type (WT) mice with ALI to assess the effect of TFPI on endothelial barrier function and inflammation. TFPI deletion markedly exacerbated LPS histopathological changes in lung, and the LPS changes in protein, EBD extravasation, proinflammatory cytokines TNF-α, IL-1β, and IL-6 in BALF in lung. The number and infiltration of white blood cells (WBCs) from BALF and lung tissue of TFPI cKO mice with LPS-challenged ALI was increased compared to WT mice with LPS-challenged ALI. We also found further increased toll-like receptor 4 and nuclear factor kappa-light-chain-enhancer of activated B cells activation and additional expression of vascular cell adhesion molecule 1 and reduction of angiotensin converting enzyme 2 expression in TFPI cKO+LPS mice compared with WT+LPS mice. Endothelial-specific TFPI deficiency promoted LPS-induced pulmonary inflammation and endothelial barrier permeability possibly via toll-like receptor 4-mediated nuclear factor kappa-light-chain-enhancer of activated B cells signaling pathway activation.

GYY4137 attenuates LPS-induced acute lung injury via heme oxygenase-1 modulation

GYY4137, a slow-releasing hydrogen sulfide (H2S) donor, has been reported to exert anti-inflammatory activity and protect against sepsis. Heme oxygenase-1 (HO-1) is an important anti-inflammatory heat shock protein and plays a similar effect on sepsis. This study investigated the role of GYY4137 in acute lung injury (ALI) via HO-1 regulation. Lung injury was assessed in mice challenged with intratracheal lipopolysaccharide (LPS) and the mechanism of anti-inflammatory effects of GYY4137 was investigated in mice and RAW264.7 cells. GYY4137 reduced the LPS-mediated pulmonary injury and neutrophil infiltration, and inhibited the LPS-induced production of proinflammatory cytokines, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression. Moreover, GYY4137 suppressed the LPS-evoked NF-κB activation in RAW264.7 cells. GYY4137, not time-expired GYY4137 significantly induced HO-1 expression compared with the LPS group. The beneficial effects of GYY4137 above were reversed by the HO-1 inhibitor tin protoporphyrin (SnPP). These results suggest an anti-inflammatory effect and a therapeutic role of GYY4137 in LPS-induced ALI via HO-1 regulation.

Intravascular heavy chain-modification of hyaluronan during endotoxic shock

During inflammation, the covalent linking of the ubiquitous extracellular polysaccharide hyaluronan (HA) with the heavy chains (HC) of the serum protein inter alpha inhibitor (IαI) is exclusively mediated by the enzyme tumor necrosis factor α (TNFα)-stimulated-gene-6 (TSG-6). While significant advances have been made regarding how HC-modified HA (HC-HA) is an important regulator of inflammation, it remains unclear why HC-HA plays a critical role in promoting survival in intraperitoneal lipopolysaccharide (LPS)-induced endotoxemia while exerting only a modest role in the outcomes following intratracheal exposure to LPS. To address this gap, the two models of intraperitoneal LPS-induced endotoxic shock and intratracheal LPS-induced acute lung injury were directly compared in TSG-6 knockout mice and littermate controls. HC-HA formation, endogenous TSG-6 activity, and inflammatory markers were assessed in plasma and lung tissue. TSG-6 knockout mice exhibited accelerated mortality during endotoxic shock. While both intraperitoneal and intratracheal LPS induced HC-HA formation in lung parenchyma, only systemically-induced endotoxemia increased plasma TSG-6 levels and intravascular HC-HA formation. Cultured human lung microvascular endothelial cells secreted TSG-6 in response to both TNFα and IL1β stimulation, indicating that, in addition to inflammatory cells, the endothelium may secrete TSG-6 into circulation during systemic inflammation. These data show for the first time that LPS-induced systemic inflammation is uniquely characterized by significant vascular induction of TSG-6 and HC-HA, which may contribute to improved outcomes of endotoxemia.

Obesity and Type 2 Diabetes mellitus induce lipopolysaccharide tolerance in rat neutrophils

Obesity and diabetes implicate in various health complications and increased mortality caused by infection. Innate immune system is broadly affected by these diseases, leading the patients to an immunosuppressive state. A mechanism that leads innate immune cells to a less capacity of killing microorganism is the impaired TLR4 activation. TLR4 recognizes a component of the outer membrane of Gram-negative bacteria, lipopolysaccharide (LPS), and when activated increases the production of inflammatory substances. Neutrophils are components of the innate immune system and are the first responders to an invading agent. The correct activation of TLR4 in these cells is required for the initiation of the inflammatory process and elimination of the microorganisms. The aim of this study was to evaluate the influence of type 2 diabetes and obesity in the TLR4 pathway in rat neutrophils. Two experimental models were used: Goto-Kakizaki rats and high-fat-diet induced obese Wistar rats. To evaluate neutrophil response to LPS, intratracheal LPS instillation was used. Neutrophils from obese and diabetic animals exhibited tolerance to LPS, mainly by the impaired production of cytokines and chemokines and the low content of phospho-NFκB and phospho-IKBα. Neutrophils from both experimental models had increased cell death, impaired in vivo migration and myeloperoxidase activity.

14 - An inhibitor of NOX2 activation prevents lung injury following LPS administration

We have identified a peptide (called PIP-2) that specifically inhibits the activation of NADPH oxidase, type 2 (NOX2). Mice (C57Bl/6) were administered intratracheal lipopolysaccharide (LPS derived from E.coli) at 5 μg/g body wt. Lungs from mice that were evaluated at 12, 16, or 24 hours after LPS showed inflammation (increased cells and protein in the lung lavage fluid), oxidative stress (increased TBARS, 8-isoprostanes, and protein carbonyls in the lung homogenate ), and lung edema (increased lung wet to dry weight ratio). These indices of acute lung injury (ALI) were stable through 24 hr with partial recovery at 48 hr after LPS. Treatment with PIP-2 at 0, 12, or 16 hr after LPS resulted in a complete resolution of the indices of lung injury when lungs were examined at 24 hr. We interpret these results as a reflection of ongoing injury and repair processes in the lung following LPS. In control LPS-treated lungs, ongoing injury and repair were essentially in balance so that the manifestations of acute lung injury remained constant between 12 and 24 hr after LPS administration. However, with administration of PIP-2 at 12 or 16 hr, inhibition of NOX2 activation allowed the lung to fully repair prior to its examination at 24 hr. Thus, inhibition of NOX2 activation can protect lungs against inflammatory lung injury associated with administration of LPS. The non-toxic PIP-2 peptide could be a useful therapeutic agent to prevent oxidative lung injury.

Roles of apoptosis and inflammation in a rat model of acute lung injury induced right ventricular dysfunction.

The effect of intratracheal lipopolysaccharide (LPS) instillation on right ventricular dysfunction in rats was studied with the aim of exploring underlying mechanisms. A single dose of LPS (10 mg/kg) or an equal volume of saline was instilled intratracheally and lung injury evaluated using histopathologic scoring and wet/dry (W/D) weight ratio at 6 or 12 h post-administration. Besides, serum atrial natriuretic peptide (ANP) was detected using an enzyme-linked immunosorbent assay (ELISA) and right ventricle β-myosin heavy chain (β-MHC) presence was examined using reverse transcription polymerase chain reaction (RT-PCR). Echocardiography examined pulmonary artery acceleration time (PAAT), right ventricular free wall thickness (RVFWT), tricuspid annular plane systolic excursion (TAPSE), and right ventricular end-diastolic diameter (RVEDD). In addition, right ventricular TUNEL staining and Western blots of Bax and Bcl-2 were performed. Right ventricle and left ventricle caspases-3, -8, and -9 activity were examined using fluorometric assay. Finally, right ventricle myeloperoxidase (MPO) neutrophil staining, and right ventricle and plasma cytokines TNF-α, IL-1β, IL-6 detection was performed. Histopathologic lung injury and increased W/D weight ratio was seen at 6 h after LPS intratracheal instillation, along with increased ANP, but not β-MHC. Pulmonary hypertension was indicated by decreased PAAT at 6 h post-exposure. Right ventricular systolic dysfunction and dilation were observed at 12 h post-exposure, as indicated by a significant decrease of TAPSE and increase of RVEDD. Of note, the procedure led to an increased right ventricle TUNEL positive cardiomyocytes, an increased Bax/Bcl-2 ratio, and increased right and left ventricle caspases-3, -8, and -9 activity as early as 6 h post-exposure, which was paralleled by increased right ventricle MPO staining and increased expression of right ventricle and serum cytokines TNF-α, IL-1β, and IL-6. As well as acute lung injury, a single dose of LPS intratracheally instilled can induce pulmonary hypertension at 6 h post-exposure, with obvious right ventricular systolic dysfunction and right ventricular dilation present at 12 h post-exposure, possibly via cardiomyocytes apoptosis and inflammation.

Investigations on anti-inflammatory and protective activity of Ethyl acetate fraction isolated from Vernonia Cinerea (L) in Spargue Dawley rats

Objective: To evaluate effect of ethyl acetate fraction isolated from Vernonia cinerea (L) (EAVC) in acute lung inflammation in rats. Methodology: Rats were pretreated with vehicle (CMC 0.5%), EAVC(25, 50 & 100 mg/kg po), roflumilast (1mg/kg po), once a day for 3 consecutive days. 2hrs after the last treatment the rats were intratracheally injected with lipopolysaccharide (LPS), (1mg/kg) while control group received saline(1ml/kg) under anesthesia. After 4hrs of intratracheal LPS injection, animals were sacrificed and BALF was collected. Lungs were perfused, dissected out and used for biochemical estimations and histological examination. Results: EAVC (50 & 100 mg/kg po) significantly (p < 0.05) inhibited MPO, total protein, albumin, total cells, neutrophils and histamine content in BALF indicating suppression of lung permeability. Similarly, MOP, catalase enzyme activity in lung tissue homogenate were reduced as compared to positive control group. Histological evaluation revealed marked reduction in perivascular infiltration and edema in drug treated animals. Conclusion: EAVC (50 & 100 mg/kg po) exhibited protective effect in LPS induced ALI in rats by inhibiting lung permeability and inflammation.

Anti-inflammatory Role of Mesenchymal Stem Cells in an Acute Lung Injury Mouse Model.

Background Mesenchymal stem cells (MSCs) attenuate injury in various lung injury models through paracrine effects. We hypothesized that intratracheal transplantation of allogenic MSCs could attenuate lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice, mediated by anti-inflammatory responses.Methods Six-week-old male mice were randomized to either the control or the ALI group. ALI was induced by intratracheal LPS instillation. Four hours after LPS instillation, MSCs or phosphate-buffered saline was randomly intratracheally administered. Neutrophil count and protein concentration in bronchoalveolar lavage fluid (BALF); lung histology; levels of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and macrophage inflammatory protein-2; and the expression of proliferation cell nuclear antigen (PCNA), caspase-3, and caspase-9 were evaluated at 48 hours after injury.Results Treatment with MSCs attenuated lung injury in ALI mice by decreasing protein level and neutrophil recruitment into the BALF and improving the histologic change. MSCs also decreased the protein levels of proinflammatory cytokines including IL-1β, IL-6, and TNF-α, but had little effect on the protein expression of PCNA, caspase-3, and caspase-9.Conclusions Intratracheal injection of bone marrow-derived allogenic MSCs attenuates LPS-induced ALI via immunomodulatory effects.

P-310 - A peptide derived from naturally occurring surfactant protein A (SP-A) inhibits the phospholipase A2 (PLA2) activity of peroxiredoxin 6 (Prdx6) and NOX2 activation and prevents mouse lung injury following intratracheal LPS

We have developed a peptide based on the naturally occurring 26 kDa SP-A protein that inhibits the PLA2 but not the GPx activity of Prdx6. A 16 aa sequence was predicted by the ZDOCK program as the molecular site for binding of SP-A to Prdx6 and was confirmed using ITC and CD measurements.The peptide (called PLA2-inhibitory peptide or PIP) inhibited the PLA2 activity of recombinant human Prdx6 by ~70% and similarly inhibited Prdx6-associated PLA2 activity in homogenates of mouse lungs while presence of the peptide prevented the activation of lung NOX2.We have found since that a 9 aa peptide, named PIP-2,retains the Prdx6-inhibitory properties of both the protein and PIP. PIP was tested in a mouse model of acute lung injury (ALI) generated by the intratracheal instillation of bacterial lipopolysaccharide (LPS).PIP was encapsulated in liposomes for intracellular delivery and administered IV to mice either co-incidentally with or at 12–14 h after LPS. Mouse lungs evaluated at 24 h after LPS showed a marked decrease of lung leukocytic infiltration, of altered permeability, and of the oxidation of tissue macromolecules in the animals receiving PIP. These studies suggest that PIP might be a useful therapeutic for the prevention and treatment of ALI.

Toll-like receptor-4-interacting surfactant protein-A-derived peptide reduces inflammation in a mouse model of intratracheal lipopolysaccharide challenge.

Abstract Acute lung injury is characterized by inflammation and respiratory failure. In this work, we evaluated the effect of Toll-like receptor 4 (TLR4)-interacting SPA4 peptide on inflammatory and physiological parameters in cells and in mice against lipopolysaccharide (LPS). Cellular toxicity was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Binding of SPA4 peptide was studied in cells transiently transfected with a plasmid DNA encoding TLR4. We assessed the changes in the levels of cytokines and chemokines in supernatants of cells treated with SPA4 peptide simultaneously or post-LPS challenge. The extent of lung edema, levels of cytokines and chemokines, and changes in lung histopathology, blood gas and electrolytes, and serum lactate levels, were then determined after SPA4 peptide treatment in a mouse model of acute lung inflammation and injury induced by intratracheal LPS challenge. Our results demonstrate that the SPA4 peptide is not toxic, and binds to TLR4 at the cellular level. A significant reduction was observed in secreted levels of LPS-induced cytokines and chemokines in cell-free supernatants. Correspondingly, the levels of TNF-α, IL-1β, IL-6, MIP-2, KC, and MCP-1 were reduced in bronchoalveolar lavage fluids of SPA4 peptide-treated mice. Moreover, SPA4 peptide treatment attenuated the LPS-induced pulmonary inflammation, lung edema, serum lactate, and blood gas and electrolyte parameters. Together, our results suggest that the SPA4 peptide treatment can help control lung inflammation and injury.

Nrf2 activation protects against intratracheal LPS induced mouse/murine acute respiratory distress syndrome by regulating macrophage polarization

The transcription factor nuclear factor E2-related factor 2 (Nrf2) is known to control the expression of antioxidant response elements and cytoprotective genes and modulate inflammatory response, helping to ameliorate damage in many diseases. Exactly how Nrf2 regulates innate inflammatory homeostasis remains unclear. In this study, we provide in vitro and in vivo evidence that Nrf2 plays a crucial role in macrophage polarization and acute respiratory distress syndrome (ARDS). We conducted in vitro experiments using a mouse alveolar macrophage cell line as well as primary cultures of macrophages in which cells were exposed to lipopolysaccharide (LPS) or interferon-γ in order to mimic ARDS, in the presence or absence of the Nrf2 activator tert-butylhydroquinone (tBHQ). Using siRNA-mediated Nrf2 knockdown, we showed that Nrf2 inhibited the inflammatory response by promoting M2 macrophage polarization and inhibiting M1 macrophage polarization. At the same time, tBHQ activated Nrf2-mediated inhibition of the p65 nuclear factor-κB pathway and activation of peroxisome proliferator-activated receptor-γ, which play important roles in regulating macrophage polarization. We also conducted in vivo experiments in which mice were given tBHQ with or without intratracheal LPS, then their survival was monitored, lung injury was assessed using histology, and levels of pro- and anti-inflammatory cytokines were assayed in the lungs and serum. Activation of Nrf2 with tBHQ dramatically reduced LPS-induced mortality and lung injury, down-regulated pro-inflammatory mediators and up-regulated anti-inflammatory mediators. These results suggest that Nrf2 can help prevent ARDS progression by promoting M2 polarization of macrophages. Interfering with Nrf2 may be an effective strategy for reprogramming macrophage polarization in order to treat ARDS.