Pneumosepsis survival in the setting of obesity leads to persistent steatohepatitis and metabolic dysfunction.

BACKGROUND: As critical care practice evolves, the sepsis survivor population continues to expand, often with lingering inflammation in many organs, including the liver. Given the concurrently increasing population of patients with NAFLD, in this study, we aimed to understand the long-term effect of sepsis on pre-existing NAFLD and hyperglycemia. METHODS: Male mice were randomized to a high-fat diet or a control diet (CD). After 24 weeks on diet, mice were inoculated with Klebsiella pneumoniae (Kpa). Serial glucose tolerance tests, and insulin and pyruvate challenge tests were performed 1 week before infection and at 2 and 6 weeks after infection. Whole tissue RNA sequencing and histological evaluation of the liver were performed. To test whether persistent inflammation could be reproduced in other abnormal liver environments, mice were also challenged with Kpa after exposure to a methionine-choline-deficient high-fat diet. Finally, a retrospective cohort of 65,139 patients was analyzed to evaluate whether obesity was associated with liver injury after sepsis. RESULTS: After Kpa inoculation, high-fat diet mice had normalized fasting blood glucose without a change in insulin sensitivity but with a notable decrease in pyruvate utilization. Liver examination revealed focal macrophage collections and a unique inflammatory gene signature on RNA analysis. In the clinical cohort, preobesity, and class 1 and class 2 obesity were associated with increased odds of elevated aminotransferase levels 1-2 years after sepsis. CONCLUSIONS: The combination of diet-induced obesity and pneumosepsis survival in a murine model resulted in unique changes in gluconeogenesis and liver inflammation, consistent with the progression of benign steatosis to steatohepatitis. In a cohort study, obese patients had an increased risk of elevated aminotransferase levels 1-2 years following sepsis.

Polysalicylic Acid Polymer Microparticle Decoys Therapeutically Treat Acute Respiratory Distress Syndrome.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) remain problematic due to high mortality rates and lack of effective treatments. Neutrophilic injury contributes to mortality in ALI/ARDS. Here, technology for rapid ARDS intervention is developed and evaluated, where intravenous salicylic acid-based polymer microparticles, i.e., Poly-Aspirin (Poly-A), interfere with neutrophils in blood, reducing lung neutrophil infiltration and injury in vivo in mouse models of ALI/ARDS. Importantly, Poly-A particles reduce multiple inflammatory cytokines in the airway and bacterial load in the bloodstream in a live bacteria lung infection model of ARDS, drastically improving survival. It is observed that phagocytosis of the Poly-A microparticles, with salicylic acid in the polymer backbone, alters the neutrophil surface expression of adhesion molecules, potentially contributing to their added therapeutic benefits. Given the proven safety profile of the microparticle degradation products-salicylic acid and adipic acid-it is anticipated that the Poly-A particles represent a therapeutic strategy in ARDS with a rare opportunity for rapid clinical translation.

Ultrasensitive Multiparameter Phenotyping of Rare Cells Using an Integrated Digital-Molecular-Counting Microfluidic Well Plate.

Integrated microfluidic cellular phenotyping platforms provide a promising means of studying a variety of inflammatory diseases mediated by cell-secreted cytokines. However, immunosensors integrated in previous microfluidic platforms lack the sensitivity to detect small signals in the cellular secretion of proinflammatory cytokines with high precision. This limitation prohibits researchers from studying cells secreting cytokines at low abundance or existing at a small population. Herein, the authors present an integrated platform named the "digital Phenoplate (dPP)," which integrates digital immunosensors into a microfluidic chip with on-chip cell assay chambers, and demonstrates ultrasensitive cellular cytokine secretory profile measurement. The integrated sensors yield a limit of detection as small as 0.25 pg mL-1 for mouse tumor necrosis factor alpha (TNF-α). Each on-chip cell assay chamber confines cells whose population ranges from ≈20 to 600 in arrayed single-cell trapping microwells. Together, these microfluidic features of the dPP simultaneously permit precise counting and image-based cytometry of individual cells while performing parallel measurements of TNF-α released from rare cells under multiple stimulant conditions for multiple samples. The dPP platform is broadly applicable to the characterization of cellular phenotypes demanding high precision and high throughput.

Long-term survivors of murine sepsis are predisposed to enhanced LPS-induced lung injury and proinflammatory immune reprogramming.

Millions of people who survive sepsis each year are rehospitalized and die due to late pulmonary complications. To prevent and treat these complications, biomarkers and molecular mediators must be identified. Persistent immune reprogramming in the form of immunoparalysis and impaired host defense is proposed to mediate late pulmonary complications after sepsis, particularly new pulmonary infections. However, immune reprogramming may also involve enhanced/primed responses to secondary stimuli, although their contribution to long-term sepsis complications remains understudied. We hypothesize that enhanced/primed immune responses in the lungs of sepsis survivors are associated with late pulmonary complications. To this end, we developed a murine sepsis model using cecal ligation and puncture (CLP) followed 3 wk later by administration of intranasal lipopolysaccharide to induce inflammatory lung injury. Mice surviving sepsis exhibit enhanced lung injury with increased alveolar permeability, neutrophil recruitment, and enhanced Ly6Chi monocyte Tnf expression. To determine the mediators of enhanced lung injury, we performed flow cytometry and RNA sequencing of lungs 3 wk after CLP, prior to lipopolysaccharide. Sepsis survivor mice showed expanded Ly6Chi monocytes populations and increased expression of many inflammatory genes. Of these, S100A8/A9 was also elevated in the circulation of human sepsis survivors for months after sepsis, validating our model and identifying S100A8/A9 as a potential biomarker and therapeutic target for long-term pulmonary complications after sepsis. These data provide new insight into the importance of enhanced/primed immune responses in survivors of sepsis and establish a foundation for additional investigation into the mechanisms mediating this response.

Thrombospondin-1 Restricts Interleukin-36γ-Mediated Neutrophilic Inflammation during Pseudomonas aeruginosa Pulmonary Infection.

Interleukin-36γ (IL-36γ), a member of the IL-1 cytokine superfamily, amplifies lung inflammation and impairs host defense during acute pulmonary Pseudomonas aeruginosa infection. To be fully active, IL-36γ is cleaved at its N-terminal region by proteases such as neutrophil elastase (NE) and cathepsin S (CatS). However, it remains unclear whether limiting extracellular proteolysis restrains the inflammatory cascade triggered by IL-36γ during P. aeruginosa infection. Thrombospondin-1 (TSP-1) is a matricellular protein with inhibitory activity against NE and the pathogen-secreted Pseudomonas elastase LasB-both proteases implicated in amplifying inflammation. We hypothesized that TSP-1 tempers the inflammatory response during lung P. aeruginosa infection by inhibiting the proteolytic environment required for IL-36γ activation. Compared to wild-type (WT) mice, TSP-1-deficient (Thbs1-/-) mice exhibited a hyperinflammatory response in the lungs during P. aeruginosa infection, with increased cytokine production and an unrestrained extracellular proteolytic environment characterized by higher free NE and LasB, but not CatS activity. LasB cleaved IL-36γ proximally to M19 at a cleavage site distinct from those generated by NE and CatS, which cleave IL-36γ proximally to Y16 and S18, respectively. N-terminal truncation experiments in silico predicted that the M19 and the S18 isoforms bind the IL-36R complex almost identically. IL-36γ neutralization ameliorated the hyperinflammatory response and improved lung immunity in Thbs1-/- mice during P. aeruginosa infection. Moreover, administration of cleaved IL-36γ induced cytokine production and neutrophil recruitment and activation that was accentuated in Thbs1-/- mice lungs. Collectively, our data show that TSP-1 regulates lung neutrophilic inflammation and facilitates host defense by restraining the extracellular proteolytic environment required for IL-36γ activation.IMPORTANCEPseudomonas aeruginosa pulmonary infection can lead to exaggerated neutrophilic inflammation and tissue destruction, yet host factors that regulate the neutrophilic response are not fully known. IL-36γ is a proinflammatory cytokine that dramatically increases in bioactivity following N-terminal processing by proteases. Here, we demonstrate that thrombospondin-1, a host matricellular protein, limits N-terminal processing of IL-36γ by neutrophil elastase and the Pseudomonas aeruginosa-secreted protease LasB. Thrombospondin-1-deficient mice (Thbs1-/-) exhibit a hyperinflammatory response following infection. Whereas IL-36γ neutralization reduces inflammatory cytokine production, limits neutrophil activation, and improves host defense in Thbs1-/- mice, cleaved IL-36γ administration amplifies neutrophilic inflammation in Thbs1-/- mice. Our findings indicate that thrombospondin-1 guards against feed-forward neutrophilic inflammation mediated by IL-36γ in the lung by restraining the extracellular proteolytic environment.

A digital protein microarray for COVID-19 cytokine storm monitoring.

Despite widespread concern regarding cytokine storms leading to severe morbidity in COVID-19, rapid cytokine assays are not routinely available for monitoring critically ill patients. We report the clinical application of a digital protein microarray platform for rapid multiplex quantification of cytokines from critically ill COVID-19 patients admitted to the intensive care unit (ICU) at the University of Michigan Hospital. The platform comprises two low-cost modules: (i) a semi-automated fluidic dispensing/mixing module that can be operated inside a biosafety cabinet to minimize the exposure of the technician to the virus infection and (ii) a 12-12-15 inch compact fluorescence optical scanner for the potential near-bedside readout. The platform enabled daily cytokine analysis in clinical practice with high sensitivity (<0.4 pg mL-1), inter-assay repeatability (∼10% CV), and rapid operation providing feedback on the progress of therapy within 4 hours. This test allowed us to perform serial monitoring of two critically ill patients with respiratory failure and to support immunomodulatory therapy using the selective cytopheretic device (SCD). We also observed clear interleukin-6 (IL-6) elevations after receiving tocilizumab (IL-6 inhibitor) while significant cytokine profile variability exists across all critically ill COVID-19 patients and to discover a weak correlation between IL-6 to clinical biomarkers, such as ferritin and C-reactive protein (CRP). Our data revealed large subject-to-subject variability in patients' response to COVID-19, reaffirming the need for a personalized strategy guided by rapid cytokine assays.

IL-36 Cytokines Promote Inflammation in the Lungs of Long-Term Smokers.

Chronic obstructive pulmonary disease (COPD) is a progressive inflammatory lung disease with high morbidity and mortality. The IL-36 family are proinflammatory cytokines that are known to shape innate immune responses, including those critical to bacterial pneumonia. The objective of this study was to determine whether IL-36 cytokines promote a proinflammatory milieu in the lungs of long-term smokers with and without COPD. Concentrations of IL-36 cytokines were measured in plasma and BAL fluid from subjects in a pilot study (n = 23) of long-term smokers with and without COPD in vivo and from a variety of lung cells (from 3-5 donors) stimulated with bacteria or cigarette smoke components in vitro. Pulmonary macrophages were stimulated with IL-36 cytokines in vitro, and chemokine and cytokine production was assessed. IL-36α and IL-36γ are produced to varying degrees in murine and human lung cells in response to bacterial stimuli and cigarette smoke components in vitro. Moreover, whereas IL-36γ production is upregulated early after cigarette smoke stimulation and wanes over time, IL-36α production requires a longer duration of exposure. IL-36α and IL-36γ are enhanced systemically and locally in long-term smokers with and without COPD, and local IL-36α concentrations display a positive correlation with declining ventilatory lung function and increasing proinflammatory cytokine concentrations. In vitro, IL-36α and IL-36γ induce proinflammatory chemokines and cytokines in a concentration-dependent fashion that requires IL-36R and MyD88. IL-36 cytokine production is altered in long-term smokers with and without COPD and contributes to shaping a proinflammatory milieu in the lungs.

A Digital Protein Microarray for COVID-19 Cytokine Storm Monitoring.

Despite widespread concern for cytokine storms leading to severe morbidity in COVID-19, rapid cytokine assays are not routinely available for monitoring critically ill patients. We report the clinical application of a machine learning-based digital protein microarray platform for rapid multiplex quantification of cytokines from critically ill COVID-19 patients admitted to the intensive care unit (ICU) at the University of Michigan Hospital. The platform comprises two low-cost modules: (i) a semi-automated fluidic dispensing/mixing module that can be operated inside a biosafety cabinet to minimize the exposure of technician to the virus infection and (ii) a 12-12-15 inch compact fluorescence optical scanner for the potential near-bedside readout. The platform enabled daily cytokine analysis in clinical practice with high sensitivity (<0.4pg/mL), inter-assay repeatability (~10% CV), and near-real-time operation with a 10 min assay incubation. A cytokine profiling test with the platform allowed us to observe clear interleukin-6 (IL-6) elevations after receiving tocilizumab (IL-6 inhibitor) while significant cytokine profile variability exists across all critically ill COVID-19 patients and to discover a weak correlation between IL-6 to clinical biomarkers, such as Ferritin and CRP. Our data revealed large subject-to-subject variability in a patient's response to anti-inflammatory treatment for COVID-19, reaffirming the need for a personalized strategy guided by rapid cytokine assays.

Overlapping Roles for Interleukin-36 Cytokines in Protective Host Defense against Murine Legionella pneumophila Pneumonia.

Legionella pneumophila causes life-threatening pneumonia culminating in acute lung injury. Innate and adaptive cytokines play an important role in host defense against L. pneumophila infection. Interleukin-36 (IL-36) cytokines are recently described members of the larger IL-1 cytokine family known to exert potent inflammatory effects. In this study, we elucidated the role for IL-36 cytokines in experimental pneumonia caused by L. pneumophila Intratracheal (i.t.) administration of L. pneumophila induced the upregulation of both IL-36α and IL-36γ mRNA and protein production in the lung. Compared to the findings for L. pneumophila-infected wild-type (WT) mice, the i.t. administration of L. pneumophila to IL-36 receptor-deficient (IL-36R-/-) mice resulted in increased mortality, a delay in lung bacterial clearance, increased L. pneumophila dissemination to extrapulmonary organs, and impaired glucose homeostasis. Impaired lung bacterial clearance in IL-36R-/- mice was associated with a significantly reduced accumulation of inflammatory cells and the decreased production of proinflammatory cytokines and chemokines. Ex vivo, reduced expression of costimulatory molecules and impaired M1 polarization were observed in alveolar macrophages isolated from infected IL-36R-/- mice compared to macrophages from WT mice. While L. pneumophila-induced mortality in IL-36α- or IL-36γ-deficient mice was not different from that in WT animals, antibody-mediated neutralization of IL-36γ in IL-36α-/- mice resulted in mortality similar to that observed in IL-36R-/- mice, indicating redundant and overlapping roles for these cytokines in experimental murine L. pneumophila pneumonia.

S100A8/A9 Drives Neuroinflammatory Priming and Protects against Anxiety-like Behavior after Sepsis.

Sepsis commonly results in acute and chronic brain dysfunction, which dramatically increases the morbidity associated with this common disease. Chronic brain dysfunction in animal models of sepsis survival is linked to persistent neuroinflammation and expression of multiple cytokines. However, we have found previously that microglia predominantly upregulate the damage associated molecule S100A8/A9 after sepsis. In this article, we show that S100A8/A9 is increased in the brains of patients who died of sepsis and that S100A8 is expressed in astrocytes and myeloid cells. Using a mouse model of sepsis survival, we show that S100A8/A9 is persistently expressed in the brain after sepsis. S100A9 expression is necessary for recruitment of neutrophils to the brain and for priming production of reactive oxygen species and TNF-α secretion in microglia and macrophages. However, despite improving these indices of chronic inflammation, S100A9 deficiency results in worsened anxiety-like behavior 2 wk after sepsis. Taken together, these results indicate that S100A8/A9 contributes to several facets of neuroinflammation in sepsis survivor mice, including granulocyte recruitment and priming of microglial-reactive oxygen species and cytokine production, and that these processes may be protective against anxiety behavior in sepsis survivors.

Bacterial Dissemination to the Brain in Sepsis.

RATIONALE: Sepsis causes brain dysfunction and neuroinflammation. It is unknown whether neuroinflammation in sepsis is initiated by dissemination of bacteria to the brain and sustained by persistent infection, or whether neuroinflammation is a sterile process resulting solely from circulating inflammatory mediators. OBJECTIVES: To determine if gut bacteria translocate to the brain during sepsis, and are associated with neuroinflammation. METHODS: Murine sepsis was induced using cecal ligation and puncture, and sepsis survivor mice were compared with sham and unoperated control animals. Brain tissue of patients who died of sepsis was compared with patients who died of noninfectious causes. Bacterial taxa were characterized by 16S ribosomal RNA gene sequencing in both murine and human brain specimens; compared among sepsis and nonsepsis groups; and correlated with levels of S100A8, a marker of neuroinflammation using permutational multivariate ANOVA. MEASUREMENTS AND MAIN RESULTS: Viable gut-associated bacteria were enriched in the brains of mice 5 days after surviving abdominal sepsis (P < 0.01), and undetectable by 14 days. The community structure of brain-associated bacteria correlated with severity of neuroinflammation (P < 0.001). Furthermore, bacterial taxa detected in brains of humans who die of sepsis were distinct from those who died of noninfectious causes (P < 0.001) and correlated with S100A8/A9 expression (P < 0.05). CONCLUSIONS: Although bacterial translocation is associated with acute neuroinflammation in murine sepsis, bacterial translocation did not result in chronic cerebral infection. Postmortem analysis of patients who die of sepsis suggests a role for bacteria in acute brain dysfunction in sepsis. Further work is needed to determine if modifying gut-associated bacterial communities modulates brain dysfunction after sepsis.

Interleukin-36γ and IL-36 receptor signaling mediate impaired host immunity and lung injury in cytotoxic Pseudomonas aeruginosa pulmonary infection: Role of prostaglandin E2.

Pseudomonas aeruginosa is a Gram-negative pathogen that can lead to severe infection associated with lung injury and high mortality. The interleukin (IL)-36 cytokines (IL-36α, IL-36ß and IL-36γ) are newly described IL-1 like family cytokines that promote inflammatory response via binding to the IL-36 receptor (IL-36R). Here we investigated the functional role of IL-36 cytokines in the modulating of innate immune response against P. aeruginosa pulmonary infection. The intratracheal administration of flagellated cytotoxic P. aeruginosa (ATCC 19660) upregulated IL-36α and IL-36γ, but not IL-36ß, in the lungs. IL-36α and IL-36γ were expressed in pulmonary macrophages (PMs) and alveolar epithelial cells in response to P. aeruginosa in vitro. Mortality after bacterial challenge in IL-36 receptor deficient (IL-36R-/-) mice and IL-36γ deficient (IL-36γ-/-) mice, but not IL-36α deficient mice, was significantly lower than that of wild type mice. Decreased mortality in IL-36R-/- mice and IL-36γ-/- mice was associated with reduction in bacterial burden in the alveolar space, bacterial dissemination, production of inflammatory cytokines and lung injury, without changes in lung leukocyte influx. Interestingly, IL-36γ enhanced the production of prostaglandin E2 (PGE2) during P. aeruginosa infection in vivo and in vitro. Treatment of PMs with recombinant IL-36γ resulted in impaired bacterial killing via PGE2 and its receptor; EP2. P. aeruginosa infected EP2 deficient mice or WT mice treated with a COX-2-specific inhibitor showed decreased bacterial burden and dissemination, but no change in lung injury. Finally, we observed an increase in IL-36γ, but not IL-36α, in the airspace and plasma of patients with P. aeruginosa-induced acute respiratory distress syndrome. Thus, IL-36γ and its receptor signal not only impaired bacterial clearance in a possible PGE2 dependent fashion but also mediated lung injury during P. aeruginosa infection.

Enrichment of the lung microbiome with gut bacteria in sepsis and the acute respiratory distress syndrome.

Sepsis and the acute respiratory distress syndrome (ARDS) are major causes of mortality without targeted therapies. Although many experimental and clinical observations have implicated gut microbiota in the pathogenesis of these diseases, culture-based studies have failed to demonstrate translocation of bacteria to the lungs in critically ill patients. Here, we report culture-independent evidence that the lung microbiome is enriched with gut bacteria both in a murine model of sepsis and in humans with established ARDS. Following experimental sepsis, lung communities were dominated by viable gut-associated bacteria. Ecological analysis identified the lower gastrointestinal tract, rather than the upper respiratory tract, as the likely source community of post-sepsis lung bacteria. In bronchoalveolar lavage fluid from humans with ARDS, gut-specific bacteria (Bacteroides spp.) were common and abundant, undetected by culture and correlated with the intensity of systemic inflammation. Alveolar TNF-α, a key mediator of alveolar inflammation in ARDS, was significantly correlated with altered lung microbiota. Our results demonstrate that the lung microbiome is enriched with gut-associated bacteria in sepsis and ARDS, potentially representing a shared mechanism of pathogenesis in these common and lethal diseases.

Cecal Ligation and Puncture Results in Long-Term Central Nervous System Myeloid Inflammation.

Survivors of sepsis often experience long-term cognitive and functional decline. Previous studies utilizing lipopolysaccharide injection and cecal ligation and puncture in rodent models of sepsis have demonstrated changes in depressive-like behavior and learning and memory after sepsis, as well as evidence of myeloid inflammation and cytokine expression in the brain, but the long-term course of neuroinflammation after sepsis remains unclear. Here, we utilize cecal ligation and puncture with greater than 80% survival as a model of sepsis. We found that sepsis survivor mice demonstrate deficits in extinction of conditioned fear, but no acquisition of fear conditioning, nearly two months after sepsis. These cognitive changes occur in the absence of neuronal loss or changes in synaptic density in the hippocampus. Sepsis also resulted in infiltration of monocytes and neutrophils into the CNS at least two weeks after sepsis in a CCR2 independent manner. Cellular inflammation is accompanied by long-term expression of pro-inflammatory cytokine and chemokine genes, including TNFα and CCR2 ligands, in whole brain homogenates. Gene expression analysis of microglia revealed that while microglia do express anti-microbial genes and damage-associated molecular pattern molecules of the S100A family of genes at least 2 weeks after sepsis, they do not express the cytokines observed in whole brain homogenates. Our results indicate that in a naturalistic model of infection, sepsis results in long-term neuroinflammation, and that this sustained inflammation is likely due to interactions among multiple cell types, including resident microglia and peripherally derived myeloid cells.

IL-36γ is secreted in microparticles and exosomes by lung macrophages in response to bacteria and bacterial components.

Interleukin-36 is a family of novel interleukin-1-like proinflammatory cytokines that are highly expressed in epithelial tissues and several myeloid-derived cell types. Like those of classic interleukin-1 cytokines, the secretion mechanisms of interleukin-36 are not well understood. Interleukin-36γ secretion in dermal epithelial cells requires adenosine 5'-triphosphate, which suggests a nonclassical mechanism of secretion. In this study, murine pulmonary macrophages and human alveolar macrophages were treated with recombinant pathogen-associated molecular patterns (intact bacteria: Klebsiella pneumoniae or Streptococcus pneumoniae). Cell lysates were analyzed for messenger ribonucleic acid by quantitative real-time polymerase chain reaction, and conditioned medium was analyzed for interleukin-36γ by enzyme-linked immunosorbent assay, with or without sonication. In addition, conditioned medium was ultracentrifuged at 25,000 g and 100,000 g, to isolate microparticles and exosomes, respectively, and interleukin-36γ protein was assessed in each fraction by Western blot analysis. Interleukin-36γ mRNA was induced in both murine and human lung macrophages by a variety of pathogen-associated molecular patterns, as well as heat-killed and live Klebsiella pneumoniae and Streptococcus pneumoniae, and induction occurred in a myeloid differentiation response gene 88-dependent manner. Secretion of interleukin-36γ protein was enhanced by adenosine 5'-triphosphate. Furthermore, extracellular interleukin-36γ protein detection was markedly enhanced by sonication to disrupt membrane-bound structures. Interleukin-36γ protein was detected by Western blot in microparticles and exosome fractions isolated by ultracentrifugation. Interleukin-36γ was induced and secreted from lung macrophages in response to Gram-negative and -positive bacterial stimulation. The results suggest that interleukin-36γ is secreted in a non-Golgi-dependent manner by lung macrophages in response to Gram-positive and -negative bacterial challenge.

A Role for Low Density Lipoprotein Receptor-Related Protein 1 in the Cellular Uptake of Tissue Plasminogen Activator in the Lungs.

PURPOSE: To gain knowledge of lung clearance mechanisms of inhaled tissue plasminogen activator (tPA). METHODS: Using an in vivo mouse model and ex vivo murine whole organ cell suspensions, we examined the capability of the lungs to utilize LRP1 receptor-mediated endocytosis (RME) for the uptake of exogenous tPA with and without an LRP1 inhibitor, receptor associated protein (RAP), and quantitatively compared it to the liver. We also used a novel imaging technique to assess the amount LRP1 in sections of mouse liver and lung. RESULTS: Following intratracheal administration, tPA concentrations in the bronchoalveolar lavage fluid (BALF) declined over time following two-compartment pharmacokinetics suggestive of a RME clearance mechanism. Ex vivo studies showed that lung and liver cells are similarly capable of tPA uptake via LRP1 RME which was reduced by ~50% by RAP. The comparable lung and liver uptake of tPA is likely due to equivalent amounts of LRP1 of which there was an abundance in the alveolar epithelium. CONCLUSIONS: Our findings indicate that LRP1 RME is a candidate clearance mechanism for inhaled tPA which has implications for the development of safe and effective dosing regimens of inhaled tPA for the treatment of plastic bronchitis and other fibrin-inflammatory airway diseases in which inhaled tPA may have utility.

Epigenetic Regulation of Tolerance to Toll-Like Receptor Ligands in Alveolar Epithelial Cells.

To protect the host against exuberant inflammation and injury responses, cells have the ability to become hyporesponsive or "tolerized" to repeated stimulation by microbial and nonmicrobial insults. The lung airspace is constantly exposed to a variety of exogenous and endogenous Toll-like receptor (TLR) ligands, yet the ability of alveolar epithelial cells (AECs) to be tolerized has yet to be examined. We hypothesize that type II AECs will develop a tolerance phenotype upon repeated TLR agonist exposure. To test this hypothesis, primary AECs isolated from the lungs of mice and a murine AEC cell line (MLE-12) were stimulated with either a vehicle control or a TLR ligand for 18 hours, washed, then restimulated with either vehicle or TLR ligand for an additional 6 hours. Tolerance was assessed by measurement of TLR ligand-stimulated chemokine production (monocyte chemoattractant protein [MCP]-1/CCL2, keratinocyte chemoattractant [KC]/CXCL1, and macrophage inflammatory protein [MIP]-2/CXCL2). Sequential treatment of primary AECs or MLE-12 cells with TLR agonists resulted in induction of either tolerance or cross-tolerance. The induction of tolerance was not due to expression of specific negative regulators of TLR signaling (interleukin-1 receptor associated kinase [IRAK]-M, Toll-interacting protein [Tollip], single Ig IL-1-related receptor [SIGIRR], or suppressor of cytokine signaling [SOCS]), inhibitory microRNAs (miRs; specifically, miR-155 and miR146a), or secretion of inhibitory or regulatory soluble mediators (prostaglandin E2, IL-10, transforming growth factor-ß, or IFN-α/ß). Moreover, inhibition of histone demethylation or DNA methylation did not prevent the development of tolerance. However, treatment of AECs with the histone deacetylase inhibitors trichostatin A or suberoylanilide hyrozamine resulted in reversal of the tolerance phenotype. These findings indicate a novel mechanism by which epigenetic modification regulates the induction of tolerance in AECs.

Microarray analysis identifies IL-1 receptor type 2 as a novel candidate biomarker in patients with acute respiratory distress syndrome.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a disease associated with a high mortality rate. The initial phase is characterized by induction of inflammatory cytokines and chemokines and influx of circulating inflammatory cells, including macrophages which play a pivotal role in the innate and adaptive immune responses to injury. Growing evidence points to phenotypic heterogeneity and plasticity between various macrophage activation states. METHODS: In this study, gene expression in alveolar macrophages and circulating leukocytes from healthy control subjects and patients with ARDS was assessed by mRNA microarray analysis. RESULTS: Both alveolar macrophages and circulating leukocytes demonstrated up-regulation of genes encoding chemotactic factors, antimicrobial peptides, chemokine receptors, and matrix metalloproteinases. Two genes, the pro-inflammatory S100A12 and the anti-inflammatory IL-1 decoy receptor IL-1R2 were significantly induced in both cell populations in ARDS patients, which was confirmed by protein quantification. Although S100A12 levels did not correlate with disease severity, there was a significant association between early plasma levels of IL-1R2 and APACHE III scores at presentation. Moreover, higher levels of IL-1R2 in plasma were observed in non-survivors as compared to survivors at later stages of ARDS. CONCLUSIONS: These results suggest a hybrid state of alveolar macrophage activation in ARDS, with features of both alternative activation and immune tolerance/deactivation.. Furthermore, we have identified a novel plasma biomarker candidate in ARDS that correlates with the severity of systemic illness and mortality.

IRAK-M promotes alternative macrophage activation and fibroproliferation in bleomycin-induced lung injury.

Idiopathic pulmonary fibrosis is a devastating lung disease characterized by inflammation and the development of excessive extracellular matrix deposition. Currently, there are only limited therapeutic intervenes to offer patients diagnosed with pulmonary fibrosis. Although previous studies focused on structural cells in promoting fibrosis, our study assessed the contribution of macrophages. Recently, TLR signaling has been identified as a regulator of pulmonary fibrosis. IL-1R-associated kinase-M (IRAK-M), a MyD88-dependent inhibitor of TLR signaling, suppresses deleterious inflammation, but may paradoxically promote fibrogenesis. Mice deficient in IRAK-M (IRAK-M(-/-)) were protected against bleomycin-induced fibrosis and displayed diminished collagen deposition in association with reduced production of IL-13 compared with wild-type (WT) control mice. Bone marrow chimera experiments indicated that IRAK-M expression by bone marrow-derived cells, rather than structural cells, promoted fibrosis. After bleomycin, WT macrophages displayed an alternatively activated phenotype, whereas IRAK-M(-/-) macrophages displayed higher expression of classically activated macrophage markers. Using an in vitro coculture system, macrophages isolated from in vivo bleomycin-challenged WT, but not IRAK-M(-/-), mice promoted increased collagen and α-smooth muscle actin expression from lung fibroblasts in an IL-13-dependent fashion. Finally, IRAK-M expression is upregulated in peripheral blood cells from idiopathic pulmonary fibrosis patients and correlated with markers of alternative macrophage activation. These data indicate expression of IRAK-M skews lung macrophages toward an alternatively activated profibrotic phenotype, which promotes collagen production, leading to the progression of experimental pulmonary fibrosis.

Redundant and cooperative interactions between TLR5 and NLRC4 in protective lung mucosal immunity against Pseudomonas aeruginosa.

Flagellin is the major structural component of flagella expressed by Pseudomonas aeruginosa (PA) and other bacteria. This protein has been shown to activate the Toll-like receptor TLR5 and the Nod-like receptor Nlrc4/Ipaf, culminating in the expression of innate cytokines and antimicrobial molecules. In this study, we tested the hypothesis that TLR5 and Nlrc4 in combination are required for maximal protective lung innate mucosal immunity against PA. To test this hypothesis, we compared innate immune responses in wild-type (WT) C57B6 mice challenged with PA intratracheally to those observed in mice genetically deficient in TLR5 (TLR5(-/-)) or Nlrc4 (Nlrc4(-/-)) alone or in combination (TLR5/Nlrc4(-/-)). As compared to WT, TLR5(-/-) and Nlrc4(-/-) mice, we observed a significant increase in mortality in TLR5/Nlrc4(-/-) mice, which was associated with a >5,000-fold increase in lung PA colony-forming units and systemic bacterial dissemination. The increased mortality observed in double-deficient mice was not attributable to differences in lung leukocyte influx or lung injury responses. Levels of biologically active IL-1ß and IL-18 were reduced in the bronchoalveolar lavage fluid from PA-infected Nlrc4(-/-) and TLR5/Nlrc4(-/-) but not TLR5(-/-) mice, indicating the requirement for Nlrc4-dependent caspase-1 activation. Similarly, decreased production of biologically active IL-1ß and activation of caspase-1 was observed in PA-stimulated pulmonary macrophages isolated from Nlrc4(-/-) and TLR5/Nlrc4(-/-) but not TLR5(-/-) mice, whereas the expression of iNOS and the production of NO were significantly reduced in cells from double-mutant but not single-mutant mice. Collectively, our findings indicate that TLR5 and Nlrc4 have both unique and redundant roles in lung antibacterial mucosal immunity, and the absence of both pathogen recognition receptors results in an increase in susceptibility to invasive lung infection.