Lipopolysaccharide Induces Alveolar Macrophage Necrosis via CD14 and the P2X7 Receptor Leading to Interleukin-1α Release.

Acute lung injury (ALI) remains a serious health issue with little improvement in our understanding of the pathophysiology and therapeutic approaches. We investigated the mechanism that lipopolysaccharide (LPS) induces early neutrophil recruitment to lungs and increases pulmonary vascular permeability during ALI. Intratracheal LPS induced release of pro-interleukin-1α (IL-1α) from necrotic alveolar macrophages (AM), which activated endothelial cells (EC) to induce vascular leakage via loss of vascular endothelial (VE)-cadherin. LPS triggered the AM purinergic receptor P2X7(R) to induce Ca(2+) influx and ATP depletion, which led to necrosis. P2X7R deficiency significantly reduced necrotic death of AM and release of pro-IL-1α into the lung. CD14 was required for LPS binding to P2X7R, as CD14 neutralization significantly diminished LPS induced necrotic death of AM and pro-IL-1α release. These results demonstrate a key role for pro-IL-1α from necrotic alveolar macrophages in LPS-mediated ALI, as a critical initiator of increased vascular permeability and early neutrophil infiltration.

Alternatively spliced myeloid differentiation protein-2 inhibits TLR4-mediated lung inflammation.

We previously identified a novel alternatively spliced isoform of human myeloid differentiation protein-2 (MD-2s) that competitively inhibits binding of MD-2 to TLR4 in vitro. In this study, we investigated the protective role of MD-2s in LPS-induced acute lung injury by delivering intratracheally an adenovirus construct that expressed MD-2s (Ad-MD-2s). After adenovirus-mediated gene transfer, MD-2s was strongly expressed in lung epithelial cells and readily detected in bronchoalveolar lavage fluid. Compared to adenovirus serotype 5 containing an empty vector lacking a transgene control mice, Ad-MD-2s delivery resulted in significantly less LPS-induced inflammation in the lungs, including less protein leakage, cell recruitment, and expression of proinflammatory cytokines and chemokines, such as IL-6, keratinocyte chemoattractant, and MIP-2. Bronchoalveolar lavage fluid from Ad-MD-2s mice transferred into lungs of naive mice before intratracheal LPS challenge diminished proinflammatory cytokine levels. As house dust mite (HDM) sensitization is dependent on TLR4 and HDM Der p 2, a structural homolog of MD-2, we also investigated the effect of MD-2s on HDM-induced allergic airway inflammation. Ad-MD-2s given before HDM sensitization significantly inhibited subsequent allergic airway inflammation after HDM challenge, including reductions in eosinophils, goblet cell hyperplasia, and IL-5 levels. Our study indicates that the alternatively spliced short isoform of human MD-2 could be a potential therapeutic candidate to treat human diseases induced or exacerbated by TLR4 signaling, such as Gram-negative bacterial endotoxin-induced lung injury and HDM-triggered allergic lung inflammation.

Mast cells play an important role in chlamydia pneumoniae lung infection by facilitating immune cell recruitment into the airway.

Mast cells are known as central players in allergy and anaphylaxis, and they play a pivotal role in host defense against certain pathogens. Chlamydia pneumoniae is an important human pathogen, but it is unclear what role mast cells play during C. pneumoniae infection. We infected C57BL/6 (wild-type [WT]) and mast cell-deficient mice (Kit(W-sh/W-sh) [Wsh]) with C. pneumoniae. Wsh mice showed improved survival compared with WT mice, with fewer cells in Wsh bronchoalveolar lavage fluid (BALF), despite similar levels of cytokines and chemokines. We also found a more rapid clearance of bacteria from the lungs of Wsh mice compared with WT mice. Cromolyn, a mast cell stabilizer, reduced BALF cells and bacterial burden similar to the levels seen in Wsh mice; conversely, Compound 48/80, a mast cell degranulator, increased the number of BALF cells and bacterial burden. Histology showed that WT lungs had diffuse inflammation, whereas Wsh mice had patchy accumulations of neutrophils and perivascular accumulations of lymphocytes. Infected Wsh mice had reduced amounts of matrix metalloprotease-9 in BALF and were resistant to epithelial integral membrane protein degradation, suggesting that barrier integrity remains intact in Wsh mice. Mast cell reconstitution in Wsh mice led to enhanced bacterial growth and normal epithelial integral membrane protein degradation, highlighting the specific role of mast cells in this model. These data suggest that mast cells play a detrimental role during C. pneumoniae infection by facilitating immune cell infiltration into the airspace and providing a more favorable replicative environment for C. pneumoniae.

The NLRP3 inflammasome is required for the development of hypoxemia in LPS/mechanical ventilation acute lung injury.

IL-1ß is a potent proinflammatory cytokine that is implicated in the pathogenesis of acute respiratory distress syndrome. We hypothesized that LPS and mechanical ventilation (MV) together could lead to IL-1ß secretion and the development of acute lung injury (ALI), and that this process would be dependent on caspase-1 and the nucleotide binding domain and leucine-rich repeat (NLR) pyrin domain containing 3 (NLRP3) inflammasome activation. The objectives of this study were to determine the specific role of IL-1ß, caspase-1, and the NLRP3 inflammasome in a two-hit model of ALI due to LPS plus MV. We used a two-hit murine model of ALI in which both inhaled LPS and MV were required for the development of hypoxemia, pulmonary neutrophil infiltration, and alveolar leakage. Nlrp3-deficent and Casp1-deficient mice had significantly diminished IL-1ß levels in bronchoalveolar lavage fluid, and were specifically protected from hypoxemia, despite similar alveolar neutrophil infiltration and leakage. The IL-1 receptor antagonist, Anakinra, significantly improved the specific development of hypoxemia without significant effects on neutrophil infiltration or alveolar leakage. MV resulted in increased bronchoalveolar lavage extracellular ATP and alveolar macrophage apoptosis as triggers of NLRP3 inflammasome activation. NLRP3 inflammasome activation and IL-1ß production play a key role in ALI caused by the combination of LPS and MV, particularly in the hypoxemia associated with acute respiratory distress syndrome. Blocking IL-1 signaling in this model specifically ameliorates hypoxemia, without affecting neutrophil infiltration and alveolar leakage, disassociating these readouts of ALI. MV causes alveolar macrophage apoptosis, a key step in the activation of NLRP3 inflammasome and production of IL-1ß.

Management of autism in the adult intensive care unit.

Autism comprises a growing segment of the population and can be a management challenge in the intensive care unit (ICU). We present the case of a 22-year-old male with severe autism and intellectual disorder who developed respiratory failure and required a prolonged ICU course. This patient exhibited severe distress, aggression, and self-injurious behavior. Management challenges included sedation, weaning from sedation, and liberation from mechanical ventilation. Success was achieved with a multispecialty team and by tailoring the environment and interactions to the patient's known preferences. The use of dexmedetomidine to wean high-dose benzodiazepines and opiates also permitted successful liberation from mechanical ventilation.

Quantitative Image Analysis at Chronic Lung Allograft Dysfunction Onset Predicts Mortality.

BACKGROUND: Chronic lung allograft dysfunction (CLAD) phenotype determines prognosis and may have therapeutic implications. Despite the clarity achieved by recent consensus statement definitions, their reliance on radiologic interpretation introduces subjectivity. The Center for Computer Vision and Imaging Biomarkers at the University of California, Los Angeles (UCLA) has established protocols for chest high-resolution computed tomography (HRCT)-based computer-aided quantification of both interstitial disease and air-trapping. We applied quantitative image analysis (QIA) at CLAD onset to demonstrate radiographic phenotypes with clinical implications. METHODS: We studied 47 first bilateral lung transplant recipients at UCLA with chest HRCT performed within 90 d of CLAD onset and 47 no-CLAD control HRCTs. QIA determined the proportion of lung volume affected by interstitial disease and air-trapping in total lung capacity and residual volume images, respectively. We compared QIA scores between no-CLAD and CLAD, and between phenotypes. We also assigned radiographic phenotypes based solely on QIA, and compared their survival outcomes. RESULTS: CLAD onset HRCTs had more lung affected by the interstitial disease (P = 0.003) than no-CLAD controls. Bronchiolitis obliterans syndrome (BOS) cases had lower scores for interstitial disease as compared with probable restrictive allograft syndrome (RAS) (P < 0.0001) and mixed CLAD (P = 0.02) phenotypes. BOS cases had more air-trapping than probable RAS (P < 0.0001). Among phenotypes assigned by QIA, the relative risk of death was greatest for mixed (relative risk [RR] 11.81), followed by RAS (RR 6.27) and BOS (RR 3.15). CONCLUSIONS: Chest HRCT QIA at CLAD onset appears promising as a method for precise determination of CLAD phenotypes with survival implications.

Novel imaging approaches for small animal models of lung disease (2017 Grover Conference series).

Imaging in small animal models of lung disease is challenging, as existing technologies are limited either by resolution or by the terminal nature of the imaging approach. Here, we describe the current state of small animal lung imaging, the technological advances of laboratory-sourced phase contrast X-ray imaging, and the application of this novel technology and its attendant image analysis techniques to the in vivo imaging of the large airways and pulmonary vasculature in murine models of lung health and disease.

Application of a novel in vivo imaging approach to measure pulmonary vascular responses in mice.

Noninvasive imaging of the murine pulmonary vasculature is challenging due to the small size of the animal, limits of resolution of the imaging technology, terminal nature of the procedure, or the need for intravenous contrast. We report the application of laboratory-based high-speed, high-resolution x-ray imaging, and image analysis to detect quantitative changes in the pulmonary vascular tree over time in the same animal without the need for intravenous contrast. Using this approach, we detected an increased number of vessels in the pulmonary vascular tree of animals after 30 min of recovery from a brief exposure to inspired gas with 10% oxygen plus 5% carbon dioxide (mean ± standard deviation: 2193 ± 382 at baseline vs. 6177 ± 1171 at 30 min of recovery; P < 0.0001). In a separate set of animals, we showed that the total pulmonary blood volume increased (P = 0.0412) while median vascular diameter decreased from 0.20 mm (IQR: 0.15-0.28 mm) to 0.18 mm (IQR: 0.14-0.26 mm; P = 0.0436) over the respiratory cycle from end-expiration to end-inspiration. These findings suggest that the noninvasive, nonintravenous contrast imaging approach reported here can detect dynamic responses of the murine pulmonary vasculature and may be a useful tool in studying these responses in models of disease.

An outbreak of Pseudomonas aeruginosa infections associated with flexible bronchoscopes.

BACKGROUND: Endoscopes, including bronchoscopes, are the medical devices most frequently associated with outbreaks of nosocomial infections. We investigated an outbreak of Pseudomonas aeruginosa infections after bronchoscopic procedures. METHODS: Microbiologic results were reviewed to determine the rates of recovery of P. aeruginosa from bronchoalveolar-lavage specimens. Environmental samples from endoscopes and the endoscopy suite were cultured. Medical records were reviewed to identify infections in the 14 days after a bronchoscopy. RESULTS: The rate of recovery of P. aeruginosa from bronchoalveolar-lavage specimens obtained with use of endoscopy-suite bronchoscopes increased from 10.4 percent at base line to 31.0 percent during the outbreak (relative risk, 2.97; 95 percent confidence interval, 2.28 to 3.90). Cultures of samples from three bronchoscopes grew P. aeruginosa, whereas cultures of samples from the environment, instrument-cleaning machines, and gastrointestinal endoscopes did not. The three bronchoscopes had been part of a nationwide recall. A total of 414 patients underwent bronchoscopy during the outbreak, and there were 48 respiratory tract and bloodstream infections among 39 of these patients (9.4 percent). In 32 infections (66.7 percent), P. aeruginosa was confirmed as a potentially causative organism. Exposure to a potentially contaminated bronchoscope may have had a role in the death of three patients. The rate of recovery of P. aeruginosa returned to base line after the instruments were removed from service. CONCLUSIONS: This large outbreak of P. aeruginosa infections related to bronchoscopy was apparently caused by a loose biopsy-port cap in the bronchoscopes. Instrument safety and surveillance methods for bronchoscopy must be improved, and better recall procedures are needed for medical devices.

Quercetin Inhibits Inflammasome Activation by Interfering with ASC Oligomerization and Prevents Interleukin-1 Mediated Mouse Vasculitis.

Interleukin-1ß (IL-1ß) is a highly inflammatory cytokine that significantly contributes to both acute and chronic inflammatory diseases. The secretion of IL-1ß requires a unique protease, caspase-1, which is activated by various protein platforms called inflammasomes. Data suggests a key role for mitochondrial reactive oxygen species for inflammasome activation. Flavonoids constitute a group of naturally occurring polyphenolic molecules with many biological activities, including antioxidant effects. In this study, we investigated the effect of three flavonoids, quercetin (QUC), naringenin, and silymarim on inflammasome activation. We found that QUC inhibits IL-1ß secretion by both the NLRP3 and AIM2 inflammasome in a dose dependent manner, but not the NLRC4 inflammasome. QUC inhibition of the inflammasome was still observed in Atg16l1 knockout macrophages, indicating that QUC's effect was autophagy independent. Since QUC inhibited both NLRP3 and AIM2 inflammasomes but not NLRC4, we assessed ASC speck formation. QUC reduced ASC speck formation and ASC oligomerization compared with controls. Additionally, QUC inhibited IL-1ß in Cryopyrin-Associated Periodic Syndromes (CAPS) macrophages, where NLRP3 inflammasome is constitutively activated. In conclusion, QUC inhibits both the NLRP3 and AIM2 inflammasome by preventing ASC oligomerization and may be a potential therapeutic candidate for Kawasaki disease vasculitis and other IL-1 mediated inflammatory diseases.

Novel analysis of 4DCT imaging quantifies progressive increases in anatomic dead space during mechanical ventilation in mice.

Increased dead space is an important prognostic marker in early acute respiratory distress syndrome (ARDS) that correlates with mortality. The cause of increased dead space in ARDS has largely been attributed to increased alveolar dead space due to ventilation/perfusion mismatching and shunt. We sought to determine whether anatomic dead space also increases in response to mechanical ventilation. Mice received intratracheal lipopolysaccharide (LPS) or saline and mechanical ventilation (MV). Four-dimensional computed tomography (4DCT) scans were performed at onset of MV and after 5 h of MV. Detailed measurements of airway volumes and lung tidal volumes were performed using image analysis software. The forced oscillation technique was used to obtain measures of airway resistance, tissue damping, and tissue elastance. The ratio of airway volumes to total tidal volume increased significantly in response to 5 h of mechanical ventilation, regardless of LPS exposure, and airways demonstrated significant variation in volumes over the respiratory cycle. These findings were associated with an increase in tissue elastance (decreased lung compliance) but without changes in tidal volumes. Airway volumes increased over time with exposure to mechanical ventilation without a concomitant increase in tidal volumes. These findings suggest that anatomic dead space fraction increases progressively with exposure to positive pressure ventilation and may represent a pathological process.NEW & NOTEWORTHY We demonstrate that anatomic dead space ventilation increases significantly over time in mice in response to mechanical ventilation. The novel functional lung-imaging techniques applied here yield sensitive measures of airway volumes that may have wide applications.

Technical Note: Contrast free angiography of the pulmonary vasculature in live mice using a laboratory x-ray source.

PURPOSE: In vivo imaging of the pulmonary vasculature in small animals is difficult yet highly desirable in order to allow study of the effects of a host of dynamic biological processes such as hypoxic pulmonary vasoconstriction. Here the authors present an approach for the quantification of changes in the vasculature. METHODS: A contrast free angiography technique is validated in silico through the use of computer-generated images and in vivo through microcomputed tomography (µCT) of live mice conducted using a laboratory-based x-ray source. Subsequent image processing on µCT data allowed for the quantification of the caliber of pulmonary vasculature without the need for external contrast agents. These measures were validated by comparing with quantitative contrast microangiography in the same mice. RESULTS: Quantification of arterial diameters from the method proposed in this study is validated against laboratory-based x-ray contrast microangiography. The authors find that there is a high degree of correlation (R = 0.91) between measures from microangiography and their contrast free method. CONCLUSIONS: A technique for quantification of murine pulmonary vasculature without the need for contrast is presented. As such, this technique could be applied for longitudinal studies of animals to study changes to vasculature without the risk of premature death in sensitive mouse models of disease. This approach may also be of value in the clinical setting.

BIG1 and BIG2, brefeldin A-inhibited guanine nucleotide-exchange factors for ADP-ribosylation factors.

BIG1 and BIG2 are large (approximately 200 kDa) guanine nucleotide-exchange proteins for ADP-ribosylation factors, or ARFs, that were isolated based on sensitivity of their guanine nucleotide-exchange activity to inhibition by brefeldin A. The intracellular distributions of BIG1 and BIG2 differ from those of other ARF guanine nucleotide-exchange proteins. In addition to its presence in Golgi membranes, BIG2 is seen in peripheral vesicular structures that most likely represent recycling endosomes, and BIG1 is found in nuclei of serum-starved HepG2 cells. Several binding partners for BIG1 and BIG2 that were identified via yeast two-hybrid screens include FKBP13 and myosin IXb for BIG1 and, for BIG2, the regulatory RIalpha subunit of protein kinase A, Exo70, and the GABA receptor beta subunit. Autosomal recessive periventricular heterotopia with microcephaly, a disorder of human embryonic development due to defective vesicular trafficking, has been attributed to mutations in BIG2. Methods for purification of BIG1 and BIG2 from HepG2 cells are presented here, along with a summary of information regarding their structure and function.

Nicotinamide exacerbates hypoxemia in ventilator-induced lung injury independent of neutrophil infiltration.

BACKGROUND: Ventilator-induced lung injury is a form of acute lung injury that develops in critically ill patients on mechanical ventilation and has a high degree of mortality. Nicotinamide phosphoribosyltransferase is an enzyme that is highly upregulated in ventilator-induced lung injury and exacerbates the injury when given exogenously. Nicotinamide (vitamin B3) directly inhibits downstream pathways activated by Nicotinamide phosphoribosyltransferase and is protective in other models of acute lung injury. METHODS: We administered nicotinamide i.p. to mice undergoing mechanical ventilation with high tidal volumes to study the effects of nicotinamide on ventilator-induced lung injury. Measures of injury included oxygen saturations and bronchoalveolar lavage neutrophil counts, protein, and cytokine levels. We also measured expression of nicotinamide phosophoribosyltransferase, and its downstream effectors Sirt1 and Cebpa, Cebpb, Cebpe. We assessed the effect of nicotinamide on the production of nitric oxide during ventilator-induced lung injury. We also studied the effects of ventilator-induced lung injury in mice deficient in C/EBPε. RESULTS: Nicotinamide treatment significantly inhibited neutrophil infiltration into the lungs during ventilator-induced lung injury, but did not affect protein leakage or cytokine production. Surprisingly, mice treated with nicotinamide developed significantly worse hypoxemia during mechanical ventilation. This effect was not linked to increases in nitric oxide production or alterations in expression of Nicotinamide phosphoribosyl transferase, Sirt1, or Cebpa and Cebpb. Cebpe mRNA levels were decreased with either nicotinamide treatment or mechanical ventilation, but mice lacking C/EBPε developed the same degree of hypoxemia and ventilator-induced lung injury as wild-type mice. CONCLUSIONS: Nicotinamide treatment during VILI inhibits neutrophil infiltration of the lungs consistent with a strong anti-inflammatory effect, but paradoxically also leads to the development of significant hypoxemia. These findings suggest that pulmonary neutrophilia is not linked to hypoxemia in ventilator-induced lung injury, and that nicotinamide exacerbates hypoxemia during VILI.

Chlamydia pneumoniae infection in mice induces chronic lung inflammation, iBALT formation, and fibrosis.

Chlamydia pneumoniae (CP) lung infection can induce chronic lung inflammation and is associated with not only acute asthma but also COPD exacerbations. However, in mouse models of CP infection, most studies have investigated specifically the acute phase of the infection and not the longer-term chronic changes in the lungs. We infected C57BL/6 mice with 5 × 10(5) CP intratracheally and monitored inflammation, cellular infiltrates and cytokine levels over time to investigate the chronic inflammatory lung changes. While bacteria numbers declined by day 28, macrophage numbers remained high through day 35. Immune cell clusters were detected as early as day 14 and persisted through day 35, and stained positive for B, T, and follicular dendritic cells, indicating these clusters were inducible bronchus associated lymphoid tissues (iBALTs). Classically activated inflammatory M1 macrophages were the predominant subtype early on while alternatively activated M2 macrophages increased later during infection. Adoptive transfer of M1 but not M2 macrophages intratracheally 1 week after infection resulted in greater lung inflammation, severe fibrosis, and increased numbers of iBALTS 35 days after infection. In summary, we show that CP lung infection in mice induces chronic inflammatory changes including iBALT formations as well as fibrosis. These observations suggest that the M1 macrophages, which are part of the normal response to clear acute C. pneumoniae lung infection, result in an enhanced acute response when present in excess numbers, with greater inflammation, tissue injury, and severe fibrosis.

A case of Kombucha tea toxicity.

INTRODUCTION: Kombucha "mushroom'' tea is touted to have medicinal properties. Here, we present a case of hyperthermia, lactic acidosis, and acute renal failure within 15 hours of Kombucha tea ingestion. CASE PRESENTATION: A 22 year old male, newly diagnosed with HIV, became short of breath and febrile to 103.0F, within twelve hours of Kombucha tea ingestion. He subsequently became combative and confused, requiring sedation and intubation for airway control. Laboratories revealed a lactate of 12.9 mmol/L, and serum creatinine of 2.1 mg/dL. DISCUSSION: Kombucha tea is black tea fermented in a yeast-bacteria medium. Several case reports exist of serious, and sometimes fatal, hepatic dysfunction and lactic acidosis within close proximity to ingestion. CONCLUSION: While Kombucha tea is considered a healthy elixir, the limited evidence currently available raises considerable concern that it may pose serious health risks. Consumption of this tea should be discouraged, as it may be associated with life-threatening lactic acidosis.

Effect of protein kinase A on accumulation of brefeldin A-inhibited guanine nucleotide-exchange protein 1 (BIG1) in HepG2 cell nuclei.

Brefeldin A-inhibited guanine nucleotide-exchange proteins, BIG1 and BIG2, are activators of ADP-ribosylation factor GTPases that are essential for regulating vesicular traffic among intracellular organelles. Biochemical analyses and immunofluorescence microscopy demonstrated BIG1 in nuclei as well as membranes and cytosol of serum-starved HepG2 cells. Within 20 min after addition of 8-Br-cAMP, BIG1 accumulated in nuclei, and this effect was blocked by protein kinase A (PKA) inhibitors H-89 and PKI, suggesting a dependence on PKA-catalyzed phosphorylation. BIG2 localization was not altered by cAMP, nor did BIG2 small interfering RNA influence nuclear accumulation of BIG1 induced by cAMP. Mutant BIG1 (S883A) in which Ala replaced Ser-883, a putative PKA phosphorylation site, did not move to the nucleus with cAMP addition, whereas replacement with Asp (S883D) resulted in nuclear accumulation of BIG1 without or with cAMP exposure, consistent with the mechanistic importance of a negative charge at that site. Mutation (712KPK714) of the nuclear localization signal inhibited BIG1 accumulation in nuclei, and PKA-catalyzed phosphorylation of S883, although necessary, was not sufficient for nuclear accumulation, as shown by the double mutation S883D/nuclear localization signal. A role for microtubules in cAMP-induced translocation of BIG1 is inferred from its inhibition by nocodazole. Thus, two more critical elements of BIG1 molecular structure were identified, as well as the potential function of microtubules in a novel PKA effect on BIG1 translocation.