Immunomodulatory and clinical effects of receptor-interacting protein kinase 1 (RIPK1) inhibitor eclitasertib (SAR443122) in patients with severe COVID-19: a phase 1b, randomized, double-blinded, placebo-controlled study.

BACKGROUND: Targeting receptor-interacting serine/threonine protein kinase 1 could mitigate the devastating sequelae of the hyperinflammatory state observed in severe cases of COVID-19. This study explored the immunomodulatory and clinical effects of the receptor-interacting serine/threonine protein kinase 1 inhibitor SAR443122 (eclitasertib) in patients with severe COVID-19. METHODS: In this Phase 1b, double-blinded, placebo-controlled study (NCT04469621) a total of 82 patients were screened, of whom 68 patients were eligible and randomized (2:1) to receive eclitasertib 600 mg (300 mg twice daily) or placebo up to 14 days. Primary outcome was relative change in C-reactive protein from baseline to Day 7. Time to clinical improvement using 7-point ordinal scale, ventilator/respiratory failure-free days, change in SpO2/FiO2 ratio, and biomarkers of severe COVID-19 were explored. RESULTS: Geometric mean ratio (point estimate [90% confidence interval]) of the relative change from baseline in C-reactive protein with eclitasertib vs. placebo on Day 7 was 0.85 (0.49-1.45; p = 0.30). Median time to 50% decrease in C-reactive protein from baseline was 3 days vs. 5 days (p = 0.056) with eclitasertib vs. placebo. Median time to ≥ 2-point improvement on 7-point clinical symptoms scale was 8 days vs. 10 days with eclitasertib vs. placebo (p = 0.38). Mean ventilator/respiratory failure-free days, change in baseline-adjusted SpO2/FiO2 ratio, and clinical biomarkers showed consistent numerical improvements with eclitasertib vs. placebo. The most frequently reported treatment-emergent adverse events were gastrointestinal disorders and condition aggravated/worsened COVID-19 pneumonia. CONCLUSIONS: Eclitasertib was well tolerated with consistent trends toward more rapid resolution of inflammatory biomarkers and clinical improvement in severe COVID-19 patients than placebo. GOV IDENTIFIER: NCT04469621, first posted on clinicaltrials.gov on July 14, 2020.

The Effects of Pregnancy on the Pulmonary Immune Response in a Mouse Model of LPS-Induced Acute Lung Injury.

OBJECTIVE: This study evaluated the effect of pregnancy on the pulmonary innate immune response in a mouse model of acute lung injury (ALI) using nebulized lipopolysaccharide (LPS). STUDY DESIGN: Pregnant (day 14) C57BL/6NCRL mice and nonpregnant controls received nebulized LPS for 15 minutes. Twenty-four hours later, mice were euthanized for tissue harvest. Analysis included blood and bronchoalveolar lavage fluid (BALF) differential cell counts, whole-lung inflammatory cytokine transcription levels by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), and whole-lung vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), and BALF albumin by western blot. Mature bone marrow neutrophils from uninjured pregnant and nonpregnant mice were examined for chemotactic response using a Boyden chamber and for cytokine response to LPS by RT-qPCR. RESULTS: In LPS-induced ALI, pregnant mice had higher BALF total cell (p < 0.001) and neutrophil counts (p < 0.001) as well as higher peripheral blood neutrophils (p < 0.01) than nonpregnant mice, but a similar increase (as compared with unexposed mice) in airspace albumin levels. Whole-lung expression of interleukin 6, tumor necrosis factor-α (TNF-α), and keratinocyte chemoattractant (CXCL1) was also similar. In vitro, marrow-derived neutrophils from pregnant and nonpregnant mice had similar chemotaxis to CXCL1 and N-formylmethionine-leucyl-phenylalanine, but neutrophils from pregnant mice expressed lower levels of TNF (p < 0.001) and CXCL1 (p < 0.01) after LPS stimulation. In uninjured mice, VCAM-1 was higher in lungs from pregnant versus nonpregnant mice (p < 0.05). CONCLUSION: In this model, pregnancy is associated with an augmented lung neutrophil response to ALI without increased capillary leak or whole-lung cytokine levels relative to the nonpregnant state. This may stem from increased peripheral blood neutrophil response and intrinsically increased expression of pulmonary vascular endothelial adhesion molecules. Differences in lung innate cell homeostasis may affect the response to inflammatory stimuli and explain severe lung disease in respiratory infection during pregnancy. KEY POINTS: · Inhalation of LPS in midgestation versus virgin mice is associated with increased neutrophilia.. · This occurs without a comparative increase in cytokine expression.. · This may be explained by pregnancy-enhanced pre-exposure expression of VCAM-1 and ICAM-1..

Update on the Features and Measurements of Experimental Acute Lung Injury in Animals: An Official American Thoracic Society Workshop Report.

Advancements in methods, technology, and our understanding of the pathobiology of lung injury have created the need to update the definition of experimental acute lung injury (ALI). We queried 50 participants with expertise in ALI and acute respiratory distress syndrome using a Delphi method composed of a series of electronic surveys and a virtual workshop. We propose that ALI presents as a "multidimensional entity" characterized by four "domains" that reflect the key pathophysiologic features and underlying biology of human acute respiratory distress syndrome. These domains are 1) histological evidence of tissue injury, 2) alteration of the alveolar-capillary barrier, 3) presence of an inflammatory response, and 4) physiologic dysfunction. For each domain, we present "relevant measurements," defined as those proposed by at least 30% of respondents. We propose that experimental ALI encompasses a continuum of models ranging from those focusing on gaining specific mechanistic insights to those primarily concerned with preclinical testing of novel therapeutics or interventions. We suggest that mechanistic studies may justifiably focus on a single domain of lung injury, but models must document alterations of at least three of the four domains to qualify as "experimental ALI." Finally, we propose that a time criterion defining "acute" in ALI remains relevant, but the actual time may vary based on the specific model and the aspect of injury being modeled. The continuum concept of ALI increases the flexibility and applicability of the definition to multiple models while increasing the likelihood of translating preclinical findings to critically ill patients.

Macrophages augment the skeletal muscle proinflammatory response through TNFα following LPS-induced acute lung injury.

Muscle may contribute to the systemic inflammatory environment during critical illness, but leukocyte interaction and cytokine influence on muscle and its response has not been fully explored in this context. Using an in vivo model of intratracheal lipopolysaccharide (IT LPS)-induced acute lung injury, we show that skeletal muscle rapidly responds with expression of proinflammatory genes, which may be explained by migration of LPS into the circulation. Treatment of mature C2C12 myotubes with LPS at a level achieved in the circulation following IT LPS elicited a proinflammatory cytokine expression profile similar to that of in vivo murine muscle following IT LPS. Stimulation with toll-like receptor (TLR) 2 and 3 agonists provoked comparable responses in C2C12 myotubes. Additionally, co-cultures of C2C12 myotubes and bone marrow-derived macrophages (BMDM) identified the capacity of macrophages to increase myotube proinflammatory gene expression, with tumor necrosis factor-α (TNFα) gene and protein expression largely attributable to BMDM. To investigate the contribution of TNFα in the synergy of the co-culture environment, C2C12 myotubes were treated with recombinant TNFα, co-cultures were established using TNF-deficient BMDM, and co-cultures were also depleted of TNFα using antibodies. To determine whether the in vitro observations were relevant in vivo, mice received intramuscular administration of LPS ± TNFα or TNFα-neutralizing antibodies and showed that TNFα is both sufficient and necessary to induce synergistic cytokine release from muscle. Taken together, these data demonstrate how skeletal muscle tissue may contribute proinflammatory cytokines following acute endotoxin injury and the potential of leukocytes to augment this response via TNFα secretion.

Bronchoalveolar fluid and plasma inflammatory biomarkers in contemporary ARDS patients.

Purpose: Bronchoalveolar fluid (BALF) and plasma biomarkers are often endpoints in early phase randomized trials (RCTs) in acute respiratory distress syndrome (ARDS). With ARDS mortality decreasing, we analyzed baseline biomarkers in samples from contemporary ARDS patients participating in a prior RCT and compared these to historical controls. Materials and methods: Ninety ARDS adult patients enrolled in the parent trial. BALF and blood were collected at baseline, day 4 ± 1, and day 8 ± 1. Interleukins-8/-6/-1ß/-1 receptor antagonist/-10; granulocyte colony stimulating factor; monocyte chemotactic protein-1; tumour necrosis factor-α; surfactant protein-D; von Willebrand factor; leukotriene B4; receptor for advanced glycosylation end products; soluble Fas ligand; and neutrophil counts were measured. Results: Compared to historical measurements, our values were generally substantially lower, despite our participants being similar to historical controls. For example, our BALF IL-8 and plasma IL-6 were notably lower than in a 1999 RCT of low tidal volume ventilation and a 2007 biomarker study, respectively. Conclusions: Baseline biomarker levels in current ARDS patients are substantially lower than 6-20 years before collection of these samples. These findings, whether from ICU care changes resulting in less inflammation or from variation in assay techniques over time, have important implications for design of future RCTs with biomarkers as endpoints.

Future Research Directions in Pneumonia. NHLBI Working Group Report.

Pneumonia is a complex pulmonary disease in need of new clinical approaches. Although triggered by a pathogen, pneumonia often results from dysregulations of host defense that likely precede infection. The coordinated activities of immune resistance and tissue resilience then dictate whether and how pneumonia progresses or resolves. Inadequate or inappropriate host responses lead to more severe outcomes such as acute respiratory distress syndrome and to organ dysfunction beyond the lungs and over extended time frames after pathogen clearance, some of which increase the risk for subsequent pneumonia. Improved understanding of such host responses will guide the development of novel approaches for preventing and curing pneumonia and for mitigating the subsequent pulmonary and extrapulmonary complications of pneumonia. The NHLBI assembled a working group of extramural investigators to prioritize avenues of host-directed pneumonia research that should yield novel approaches for interrupting the cycle of unhealthy decline caused by pneumonia. This report summarizes the working group's specific recommendations in the areas of pneumonia susceptibility, host response, and consequences. Overarching goals include the development of more host-focused clinical approaches for preventing and treating pneumonia, the generation of predictive tools (for pneumonia occurrence, severity, and outcome), and the elucidation of mechanisms mediating immune resistance and tissue resilience in the lung. Specific areas of research are highlighted as especially promising for making advances against pneumonia.

Obese asthmatic patients have decreased surfactant protein A levels: Mechanisms and implications.

BACKGROUND: Eosinophils are prominent in some patients with asthma and are increased in the submucosa in a subgroup of obese patients with asthma (OAs). Surfactant protein A (SP-A) modulates host responses to infectious and environmental insults. OBJECTIVE: We sought to determine whether SP-A levels are altered in OAs compared with a control group and to determine the implications of these alterations in SP-A levels in asthmatic patients. METHODS: Bronchoalveolar lavage fluid from 23 lean, 12 overweight, and 20 obese subjects were examined for SP-A. Mouse tracheal epithelial cells grown at an air-liquid interface were used for mechanistic studies. SP-A-/- mice were challenged in allergen models, and exogenous SP-A therapy was given after the last challenge. Eosinophils were visualized and quantitated in lung parenchyma by means of immunostaining. RESULTS: Significantly less SP-A (P = .002) was detected in samples from OAs compared with those from control subjects. A univariable regression model found SP-A levels were significantly negatively correlated with body mass index (r = -0.33, P = .014), whereas multivariable modeling demonstrated that the correlation depended both on asthma status (P = .017) and the interaction of asthma and body mass index (P = .008). Addition of exogenous TNF-α to mouse tracheal epithelial cells was sufficient to attenuate SP-A and eotaxin secretion. Allergen-challenged SP-A-/- mice that received SP-A therapy had significantly less tissue eosinophilia compared with mice receiving vehicle. CONCLUSIONS: SP-A functions as an important mediator in resolving tissue and lavage fluid eosinophilia in allergic mouse models. Decreased levels of SP-A in OAs, which could be due to increased local TNF-α levels, might lead to impaired eosinophil resolution and could contribute to the eosinophilic asthma phenotype.

Pulmonary Surfactant Promotes Virulence Gene Expression and Biofilm Formation in Klebsiella pneumoniae.

The interactions between Klebsiella pneumoniae and the host environment at the site of infection are largely unknown. Pulmonary surfactant serves as an initial point of contact for inhaled bacteria entering the lung and is thought to contain molecular cues that aid colonization and pathogenesis. To gain insight into this ecological transition, we characterized the transcriptional response of K. pneumoniae MGH 78578 to purified pulmonary surfactant. This work revealed changes within the K. pneumoniae transcriptome that likely contribute to host colonization, adaptation, and virulence in vivo Notable transcripts expressed under these conditions include genes involved in capsule synthesis, lipopolysaccharide modification, antibiotic resistance, biofilm formation, and metabolism. In addition, we tested the contributions of other surfactant-induced transcripts to K. pneumoniae survival using engineered isogenic KPPR1 deletion strains in a murine model of acute pneumonia. In these infection studies, we identified the MdtJI polyamine efflux pump and the ProU glycine betaine ABC transporter to be significant mediators of K. pneumoniae survival within the lung and confirmed previous evidence for the importance of de novo leucine synthesis to bacterial survival during infection. Finally, we determined that pulmonary surfactant promoted type 3 fimbria-mediated biofilm formation in K. pneumoniae and identified two surfactant constituents, phosphatidylcholine and cholesterol, that drive this response. This study provides novel insight into the interactions occurring between K. pneumoniae and the host at an important infection site and demonstrates the utility of purified lung surfactant preparations for dissecting host-lung pathogen interactions in vitro.

Mouse Modeling of Obese Lung Disease. Insights and Caveats.

As the obesity epidemic has worsened, its impact on lung health and disease has become progressively evident. The interactions between obesity and the accompanying metabolic syndrome and diseases such as asthma, pneumonia, and acute respiratory distress syndrome (ARDS) have proven complex and often counterintuitive in human studies. Hence, there is a growing need for relevant experimental approaches to understand the interactions between obesity and the lung. To this end, researchers have increasingly exploited mouse models combining both obesity and lung diseases, including ARDS, pneumonia, and asthma. Such models have both complemented and advanced the understanding we have gained from clinical studies and have allowed elegant dissections of obesity's effects on the pathogenesis of lung disease. Yet these models come with several critically important caveats that we must reflect on when interpreting their results.

A Comparative Study of Lung Host Defense in Murine Obesity Models. Insights into Neutrophil Function.

We have shown that obesity-associated attenuation of murine acute lung injury is driven, in part, by blunted neutrophil chemotaxis, yet differences were noted between the two models of obesity studied. We hypothesized that obesity-associated impairment of multiple neutrophil functions contributes to increased risk for respiratory infection but that such impairments may vary between murine models of obesity. We examined the most commonly used murine obesity models (diet-induced obesity, db/db, CPE(fat/fat), and ob/ob) using a Klebsiella pneumoniae pneumonia model and LPS-induced pneumonitis. Marrow-derived neutrophils from uninjured lean and obese mice were examined for in vitro functional responses. All obesity models showed impaired clearance of K. pneumoniae, but in differing temporal patterns. Failure to contain infection in obese mice was seen in the db/db model at both 24 and 48 hours, yet this defect was only evident at 24 hours in CPE(fat/fat) and ob/ob models, and at 48 hours in diet-induced obesity. LPS-induced airspace neutrophilia was decreased in all models, and associated with blood neutropenia in the ob/ob model but with leukocytosis in the others. Obese mouse neutrophils from all models demonstrated impaired chemotaxis, whereas neutrophil granulocyte colony-stimulating factor-mediated survival, LPS-induced cytokine transcription, and mitogen-activated protein kinase and signal transducer and activator of transcription 3 activation in response to LPS and granulocyte colony-stimulating factor, respectively, were variably impaired across the four models. Obesity-associated impairment of host response to lung infection is characterized by defects in neutrophil recruitment and survival. However, critical differences exist between commonly used mouse models of obesity and may reflect variable penetrance of elements of the metabolic syndrome, as well as other factors.

Weight Loss Decreases Inherent and Allergic Methacholine Hyperresponsiveness in Mouse Models of Diet-Induced Obese Asthma.

Obese asthma presents with inherent hyperresponsiveness to methacholine or augmented allergen-driven allergic asthma, with an even greater magnitude of methacholine hyperresponsiveness. These physiologic parameters and accompanying obese asthma symptoms can be reduced by successful weight loss, yet the underlying mechanisms remain incompletely understood. We implemented mouse models of diet-induced obesity, dietary and surgical weight loss, and environmental allergen exposure to examine the mechanisms and mediators of inherent and allergic obese asthma. We report that the methacholine hyperresponsiveness in these models of inherent obese asthma and obese allergic asthma manifests in distinct anatomical compartments but that both are amenable to interventions that induce substantial weight loss. The inherent obese asthma phenotype, with characteristic increases in distal airspace tissue resistance and tissue elastance, is associated with elevated proinflammatory cytokines that are reduced with dietary weight loss. Surprisingly, bariatric surgery-induced weight loss further elevates these cytokines while reducing methacholine responsiveness to levels similar to those in lean mice or in formerly obese mice rendered lean through dietary intervention. In contrast, the obese allergic asthma phenotype, with characteristic increases in central airway resistance, is not associated with increased adaptive immune responses, yet diet-induced weight loss reduces methacholine hyperresponsiveness without altering immunological variables. Diet-induced weight loss is effective in models of both inherent and allergic obese asthma, and our examination of the fecal microbiome revealed that the obesogenic Firmicutes/Bacteroidetes ratio was normalized after diet-induced weight loss. Our results suggest that structural, immunological, and microbiological factors contribute to the manifold presentations of obese asthma.

Myeloid derived hypoxia inducible factor 1-alpha is required for protection against pulmonary Aspergillus fumigatus infection.

Hypoxia inducible factor 1α (HIF1α) is the mammalian transcriptional factor that controls metabolism, survival, and innate immunity in response to inflammation and low oxygen. Previous work established that generation of hypoxic microenvironments occurs within the lung during infection with the human fungal pathogen Aspergillus fumigatus. Here we demonstrate that A. fumigatus stabilizes HIF1α protein early after pulmonary challenge that is inhibited by treatment of mice with the steroid triamcinolone. Utilizing myeloid deficient HIF1α mice, we observed that HIF1α is required for survival and fungal clearance early following pulmonary challenge with A. fumigatus. Unlike previously reported research with bacterial pathogens, HIF1α deficient neutrophils and macrophages were surprisingly not defective in fungal conidial killing. The increase in susceptibility of the myeloid deficient HIF1α mice to A. fumigatus was in part due to decreased early production of the chemokine CXCL1 (KC) and increased neutrophil apoptosis at the site of infection, resulting in decreased neutrophil numbers in the lung. Addition of recombinant CXCL1 restored neutrophil survival and numbers, murine survival, and fungal clearance. These results suggest that there are unique HIF1α mediated mechanisms employed by the host for protection and defense against fungal pathogen growth and invasion in the lung. Additionally, this work supports the strategy of exploring HIF1α as a therapeutic target in specific immunosuppressed populations with fungal infections.

Glutaredoxin-1 attenuates S-glutathionylation of the death receptor fas and decreases resolution of Pseudomonas aeruginosa pneumonia.

RATIONALE: The death receptor Fas is critical for bacterial clearance and survival of mice after Pseudomonas aeruginosa infection. OBJECTIVES: Fas ligand (FasL)-induced apoptosis is augmented by S-glutathionylation of Fas (Fas-SSG), which can be reversed by glutaredoxin-1 (Grx1). Therefore, the objective of this study was to investigate the interplay between Grx1 and Fas in regulating the clearance of P. aeruginosa infection. METHODS: Lung samples from patients with bronchopneumonia were analyzed by immunofluorescence. Primary tracheal epithelial cells, mice lacking the gene for Grx1 (Glrx1(-/-)), Glrx1(-/-) mice treated with caspase inhibitor, or transgenic mice overexpressing Grx1 in the airway epithelium were analyzed after infection with P. aeruginosa. MEASUREMENTS AND MAIN RESULTS: Patient lung samples positive for P. aeruginosa infection demonstrated increased Fas-SSG compared with normal lung samples. Compared with wild-type primary lung epithelial cells, infection of Glrx1(-/-) cells with P. aeruginosa showed enhanced caspase 8 and 3 activities and cell death in association with increases in Fas-SSG. Infection of Glrx1(-/-) mice with P. aeruginosa resulted in enhanced caspase activity and increased Fas-SSG as compared with wild-type littermates. Absence of Glrx1 significantly enhanced bacterial clearance, and decreased mortality postinfection with P. aeruginosa. Inhibition of caspases significantly decreased bacterial clearance postinfection with P. aeruginosa, in association with decreased Fas-SSG. In contrast, transgenic mice that overexpress Grx1 in lung epithelial cells had significantly higher lung bacterial loads, enhanced mortality, decreased caspase activation, and Fas-SSG in the lung after infection with P. aeruginosa, compared with wild-type control animals. CONCLUSIONS: These results suggest that S-glutathionylation of Fas within the lung epithelium enhances epithelial apoptosis and promotes clearance of P. aeruginosa and that glutaredoxin-1 impairs bacterial clearance and increases the severity of pneumonia in association with deglutathionylation of Fas.

The role of leptin in the development of pulmonary neutrophilia in infection and acute lung injury.

OBJECTIVES: One of the hallmarks of severe pneumonia and associated acute lung injury is neutrophil recruitment to the lung. Leptin is thought to be up-regulated in the lung following injury and to exert diverse effects on leukocytes, influencing both chemotaxis and survival. We hypothesized that pulmonary leptin contributes directly to the development of pulmonary neutrophilia during pneumonia and acute lung injury. DESIGN: Controlled human and murine in vivo and ex vivo experimental studies. SETTING: Research laboratory of a university hospital. SUBJECTS: Healthy human volunteers and subjects hospitalized with bacterial and H1N1 pneumonia. C57Bl/6 and db/db mice were also used. INTERVENTIONS: Lung samples from patients and mice with either bacterial or H1N1 pneumonia and associated acute lung injury were immunostained for leptin. Human bronchoalveolar lavage samples obtained after lipopolysaccharide-induced lung injury were assayed for leptin. C57Bl/6 mice were examined after oropharyngeal aspiration of recombinant leptin alone or in combination with Escherichia coli- or Klebsiella pneumoniae-induced pneumonia. Leptin-resistant (db/db) mice were also examined using the E. coli model. Bronchoalveolar lavage neutrophilia and cytokine levels were measured. Leptin-induced chemotaxis was examined in human blood- and murine marrow-derived neutrophils in vitro. MEASUREMENTS AND MAIN RESULTS: Injured human and murine lung tissue showed leptin induction compared to normal lung, as did human bronchoalveolar lavage following lipopolysaccharide instillation. Bronchoalveolar lavage neutrophilia in uninjured and infected mice was increased and lung bacterial load decreased by airway leptin administration, whereas bronchoalveolar lavage neutrophilia in infected leptin-resistant mice was decreased. In sterile lung injury by lipopolysaccharide, leptin also appeared to decrease airspace neutrophil apoptosis. Both human and murine neutrophils migrated toward leptin in vitro, and this required intact signaling through the Janus Kinase 2/phosphatidylinositol-4,5-bisphosphate 3-kinase pathway. CONCLUSIONS: We demonstrate that pulmonary leptin is induced in injured human and murine lungs and that this cytokine is effective in driving alveolar airspace neutrophilia. This action appears to be caused by direct effects of leptin on neutrophils.

Obesity and pro-inflammatory mediators are associated with acute kidney injury in patients with A/H1N1 influenza and acute respiratory distress syndrome.

BACKGROUND: The obesity has been shown to increase the severity of A/H1N1 infection and the development of acute respiratory distress syndrome (ARDS) and organ involvement. METHODS: Circulating levels of C-peptide, insulin, glucagon, leptin, acute phase reactants (procalcitonin, C-reactive protein, tissue plasminogen activator, and serum amyloids A and P), were measured in samples from 32 critically ill patients with A/H1N1 virus infection, 17 of whom had ARDS complicated by acute kidney injury (AKI) and 15 of whom had ARDS but did not develop AKI. RESULTS: Patients with ARDS and AKI (ARDS/AKI) had higher BMI and higher levels of C-peptide, insulin, leptin, procalcitonin and serum amyloid A compared to those ARDS patient who did not develop AKI. Adjusting for confounding variables using logistic regression analysis, higher levels of C-peptide (>0.75 ng/mL) (OR=64.8, 95% CI = 2.1-1980, p = 0.0006) and BMI>30 Kg/m(2) (OR = 42.0, 95% CI = 1.2-1478, p = 0.04) were significantly associated with the development of AKI in ARDS patients. CONCLUSION: High levels of C-peptide and BMI>30 kg/m(2) were associated with the development of AKI in ARDS patients due to A/H1N1 infection. These metabolic/obesity indicators, together with the profiles of pro-inflammatory acute phase proteins, may be important links between obesity and poor outcomes in A/H1N1 09 infection.

Interleukin-1 receptor and caspase-1 are required for the Th17 response in nitrogen dioxide-promoted allergic airway disease.

Nitrogen dioxide (NO2) is an environmental pollutant and endogenously generated oxidant associated with the development, severity, and exacerbation of asthma. NO2 exposure is capable of allergically sensitizing mice to the innocuous inhaled antigen ovalbumin (OVA), promoting neutrophil and eosinophil recruitment, and a mixed Th2/Th17 response upon antigen challenge that is reminiscent of severe asthma. However, the identity of IL-17A-producing cells and the mechanisms governing their ontogeny in NO2-promoted allergic airway disease remain unstudied. We measured the kinetics of lung inflammation after antigen challenge in NO2-promoted allergic airway disease, including inflammatory cells in bronchoalveolar lavage and antigen-specific IL-17A production from the lung. We determined that IL-17A(+) cells were predominately CD4(+)T cell receptor (TCR)ß(+) Th17 cells, and that a functional IL-1 receptor was required for Th17, but not Th2, cytokine production after in vitro antigen restimulation of lung cells. The absence of natural killer T cells, γδ T cells, or the inflammasome scaffold nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain (Nlrp)3 did not affect the development of NO2-promoted allergic inflammation or IL-17A production. Similarly, neutrophil depletion or the neutralization of IL-1α during sensitization exerted no effect on these parameters. However, the absence of caspase-1 significantly reduced IL-17A production from lung cells without affecting Th2 cytokines or lung inflammation. Finally, the intranasal administration of IL-1ß and the inhalation of antigen promoted allergic sensitization that was reflected by neutrophilic airway inflammation and IL-17A production from CD4(+)TCRß(+) Th17 cells subsequent to antigen challenge. These data implicate a role for caspase-1 and IL-1ß in the IL-1 receptor-dependent Th17 response manifest in NO2-promoted allergic airway disease.

Seasonal and pandemic influenza H1N1 viruses induce differential expression of SOCS-1 and RIG-I genes and cytokine/chemokine production in macrophages.

BACKGROUND: Infection with pandemic (pdm) A/H1N1 virus induces high levels of pro-inflammatory mediators in blood and lungs of experimental animals and humans. METHODS: To compare the involvement of seasonal A/PR/8/34 and pdm A/H1N1 virus strains in the regulation of inflammatory responses, we analyzed the changes in the whole-genome expression induced by these strains in macrophages and A549 epithelial cells. We also focused on the functional implications (cytokine production) of the differential induction of suppressors of cytokine signaling (SOCS)-1, SOCS-3, retinoid-inducible gene (RIG)-I and interferon receptor 1 (IFNAR1) genes by these viral strains in early stages of the infection. RESULTS: We identified 130 genes differentially expressed by pdm A/H1N1 and A/PR/8/34 infections in macrophages. mRNA levels of SOCS-1 and RIG-I were up-regulated in macrophages infected with the A/PR/8/34 but not with pdm A/H1N1 virus. mRNA levels of SOCS-3 and IFNAR1 induced by A/PR/8/34 and pdm A/H1N1 strains in macrophages, as well as in A549 cells were similar. We found higher levels of IL-6, TNF-α, IL-10, CCL3, CCL5, CCL4 and CXCL8 (p < 0.05) in supernatants from cultures of macrophages infected with the pdm A/H1N1 virus compared to those infected with the A/PR/8/34 strain, coincident with the lack of SOCS-1 and RIG-I expression. In contrast, levels of INF-α were higher in cultures of macrophages 48h after infection with the A/PR/8/34 strain than with the pdm A/H1N1 virus. CONCLUSIONS: These findings suggest that factors inherent to the pdm A/H1N1 viral strain may increase the production of inflammatory mediators by inhibiting SOCS-1 and modifying the expression of antiviral immunity-related genes, including RIG-I, in human macrophages.

Effects of acute and chronic low density lipoprotein exposure on neutrophil function.

Mounting evidence suggests that obesity and the metabolic syndrome have significant but often divergent effects on the innate immune system. These effects have been best established in monocytes and macrophages, particularly as a consequence of the hypercholesterolemic state. We have recently described defects in neutrophil function in the setting of both obesity and hypercholesterolemia, and hypothesized that exposure to elevated levels of lipoproteins, particularly LDL its oxidized forms, contributed to these defects. As a model of chronic cholesterol exposure, we examined functional responses of bone marrow neutrophils isolated from non-obese mice with diet-induced hypercholesterolemia compared to normal cholesterol controls. Chemotaxis, calcium flux, CD11b display, and F-actin polymerization were assayed in response to several chemoattractants, while neutrophil cytokine transcriptional response was determined to LPS. Following this, the acute effects of isolated LDL and its oxidized forms on normal neutrophils were assayed using the same functional assays. We found that neutrophils from non-obese hypercholesterolemic mice had blunted chemotaxis, altered calcium flux, and normal to augmented CD11b display with prolonged actin polymerization in response to stimuli. In response to acute exposure to lipoproteins, neutrophils showed chemotaxis to LDL which increased with the degree of LDL oxidation. Paradoxically, LDL oxidation yielded the opposite effect on LDL-induced CD11b display and actin polymerization, and both native and oxidized LDL were found to induce neutrophil transcription of the monocyte chemoattractant MCP-1. Together these findings suggest that chronic hypercholesterolemia impairs neutrophil functional responses, and these defects may be in part due to protracted signaling responses to LDL and its oxidized forms.

Leptin as regulator of pulmonary immune responses: involvement in respiratory diseases.

Leptin is an adipocyte-derived hormone, recognized as a critical mediator of the balance between food intake and energy expenditure by signalling through its functional receptor (Ob-Rb) in the hypothalamus. Structurally, leptin belongs to the long-chain helical cytokine family, and is now known to have pleiotropic functions in both innate and adaptive immunity. The presence of the functional leptin receptor in the lung together with evidence of increased airspace leptin levels arising during pulmonary inflammation, suggests an important role for leptin in lung development, respiratory immune responses and eventually pathogenesis of inflammatory respiratory diseases. The purpose of this article is to review our current understanding of leptin and its functional role on the different resident cell types of the lung in health as well as in the context of three major respiratory conditions being chronic obstructive pulmonary disease (COPD), asthma, and pneumonia.