Regulation of Versican Expression in Macrophages is Mediated by Canonical Type I Interferon Signaling via ISGF3.

Growing evidence supports a role for versican as an important component of the inflammatory response, with both pro- and anti-inflammatory roles depending on the specific context of the system or disease under investigation. Our goal is to understand the regulation of macrophage-derived versican and the role it plays in innate immunity. In previous work, we showed that LPS triggers a signaling cascade involving TLR4, the Trif adaptor, type I interferons, and the type I interferon receptor, leading to increased versican expression by macrophages. In the present study, we used a combination of chromatin immunoprecipitation, siRNA, chemical inhibitors, and mouse model approaches to investigate the regulatory events downstream of the type I interferon receptor to better define the mechanism controlling versican expression. Results indicate that transcriptional regulation by canonical type I interferon signaling via the heterotrimeric transcription factor, ISGF3, controls versican expression in macrophages exposed to LPS. This pathway is not dependent on MAPK signaling, which has been shown to regulate versican expression in other cell types. The stability of versican mRNA may also contribute to prolonged versican expression in macrophages. These findings strongly support a role for macrophage-derived versican as a type I interferon-stimulated gene and further our understanding of versican's role in regulating inflammation.

MEK1/2 inhibition decreases pro-inflammatory responses in macrophages from people with cystic fibrosis and mitigates severity of illness in experimental murine methicillin-resistant Staphylococcus aureus infection.

Chronic pulmonary bacterial infections and associated inflammation remain a cause of morbidity and mortality in people with cystic fibrosis (PwCF) despite new modulator therapies. Therapies targeting host factors that dampen detrimental inflammation without suppressing immune responses critical for controlling infections remain limited, while the development of lung infections caused by antimicrobial resistant bacteria is an increasing global problem, and a significant challenge in CF. Pharmacological compounds targeting the mammalian MAPK proteins MEK1 and MEK2, referred to as MEK1/2 inhibitor compounds, have potential combined anti-microbial and anti-inflammatory effects. Here we examined the immunomodulatory properties of MEK1/2 inhibitor compounds PD0325901, trametinib, and CI-1040 on CF innate immune cells. Human CF macrophage and neutrophil phagocytic functions were assessed by quantifying phagocytosis of serum opsonized pHrodo red E. coli, Staphylococcus aureus, and zymosan bioparticles. MEK1/2 inhibitor compounds reduced CF macrophage pro-inflammatory cytokine production without impairing CF macrophage or neutrophil phagocytic abilities. Wild-type C57BL6/J and Cftr tm1kth (F508del homozygous) mice were used to evaluate the in vivo therapeutic potential of PD0325901 compared to vehicle treatment in an intranasal methicillin-resistant Staphylococcus aureus (MRSA) infection with the community-acquired MRSA strain USA300. In both wild-type and CF mice, PD0325901 reduced inflammation associated body mass loss. Wild-type mice treated with PD0325901 had significant reduction in neutrophil-mediated inflammation compared to vehicle treatment groups, with preserved clearance of bacteria in lung, liver, or spleen 1 day after infection in either wild-type or CF mouse models. In summary, this study provides the first data evaluating the therapeutic potential of MEK1/2 inhibitor to modulate CF immune cells and demonstrates that MEK1/2 inhibitors diminish pro-inflammatory responses without impairing host defense mechanisms required for acute pathogen clearance.

Intravascular Leukocyte Labeling Refines the Distribution of Myeloid Cells in the Lung in Models of Allergen-induced Airway Inflammation.

Innate immune cell populations are critical in asthma with different functional characteristics based on tissue location, which has amplified the importance of characterizing the precise number and location of innate immune populations in murine models of asthma. In this study, we performed premortem intravascular (IV) labeling of leukocytes in mice in two models of asthma to differentiate innate immune cell populations within the IV compartment versus those residing in the lung tissue or airway lumen. We performed spectral flow cytometry analysis of the blood, suspensions of digested lung tissue, and bronchoalveolar lavage fluid. We discovered that IV labeled leukocytes do not contaminate analysis of bronchoalveolar lavage fluid but represent a significant proportion of cells in digested lung tissue. Exclusion of IV leukocytes significantly improved the accuracy of the assessments of myeloid cells in the lung tissue and provided important insights into ongoing trafficking in both eosinophilic and neutrophilic asthma models.

MEK1/2 inhibition decreases pro-inflammatory responses in macrophages from people with cystic fibrosis and mitigates severity of illness in experimental murine methicillin-resistant Staphylococcus aureus infection.

Chronic pulmonary bacterial infections and associated inflammation remain a cause of morbidity and mortality in people with cystic fibrosis (PwCF) despite new modulator therapies. Therapies targeting host factors that dampen detrimental inflammation without suppressing immune responses critical for controlling infections remain limited, while the acquisition of antibiotic resistance bacterial infections is an increasing global problem, and a significant challenge in CF. Pharmacological compounds targeting the mammalian MAPK proteins MEK1 and MEK2, referred to as MEK1/2 inhibitor compounds, have potential combined anti-microbial and anti-inflammatory effects. Here we examined the immunomodulatory properties of MEK1/2 inhibitor compounds PD0325901, trametinib, and CI-1040 on CF innate immune cells. Human CF macrophage and neutrophil phagocytic functions were assessed by quantifying phagocytosis of serum opsonized pHrodo red E. coli , Staphylococcus aureus , and zymosan bioparticles. MEK1/2 inhibitor compounds reduced CF macrophage pro-inflammatory cytokine production without impairing CF macrophage or neutrophil phagocytic abilities. Wild-type C57BL6/J and Cftr tm1kth (F508del homozygous) mice were used to evaluate the in vivo therapeutic potential of PD0325901 compared to vehicle treatment in an intranasal methicillin-resistant Staphylococcus aureus (MRSA) infection with the community-acquired MRSA strain USA300. In both wild-type and CF mice, PD0325901 reduced infection related weight loss compared to vehicle treatment groups but did not impair clearance of bacteria in lung, liver, or spleen 1 day after infection. In summary, this study provides the first data evaluating the therapeutic potential of MEK1/2 inhibitor to modulate CF immune cells, and demonstrates that MEK1/2 inhibitors dampen pro-inflammatory responses without impairing host defense mechanisms mediating pathogen clearance.

MAP2K2 Delays Recovery in Murine Models of Acute Lung Injury and Associates with Acute Respiratory Distress Syndrome Outcome.

Acute respiratory distress syndrome (ARDS) remains a significant problem in need of new pharmaceutical approaches to improve its resolution. Studies comparing gene expression signatures in rodents and humans with lung injury reveal conserved pathways, including MAPK (mitogen-activated protein kinase)/ERK (extracellular signal-related protein kinase) activation. In preclinical acute lung injury (ALI) models, inhibition of MAP2K1 (MAPK kinase 1)/MAP2K2 (MAPK kinase 2) improves measures of ALI. Myeloid cell deletion of MAP2K1 results in sustained MAP2K2 activation and nonresolving ALI, suggesting that MAP2K2 deactivation may be a key driver of ALI resolution. We used human genomic data from the iSPAAR (Identification of SNPs Predisposing to Altered Acute Lung Injury Risk) Consortium to assess genetic variants in MAP2K1 and MAP2K2 for association with mortality from ARDS. To determine the role of MAP2K2 in ALI recovery, we studied mice deficient in Map2k2 (Mek2-/-) and wild-type control mice in ALI models. We identified a MAP2K2 variant that was associated with death in ARDS and MAP2K2 expression. In Pseudomonas aeruginosa ALI, Mek2-/- mice had similar early alveolar neutrophilic recruitment but faster resolution of alveolar neutrophilia and vascular leak. Gene expression analysis revealed a role for MAP2K2 in promoting and sustaining select proinflammatory pathway activation in ALI. Bone marrow chimera studies indicate that leukocyte MAP2K2 is the key regulator of ALI duration. These studies implicate a role for MAP2K2 in ALI duration via transcriptional regulation of inflammatory programming with potential relevance to ARDS. Targeting leukocyte MAP2K2 may be an effective strategy to promote ALI resolution.

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.

Evaluation of Nutritional Gel Supplementation in C57BL/6J Mice Infected with Mouse-Adapted Influenza A/PR/8/34 Virus.

Mice are a common animal model for the study of influenza virus A (IAV). IAV infection causes weight loss due to anorexia and dehydration, which can result in early removal of mice from a study when they reach a humane endpoint. To reduce the number of mice prematurely removed from an experiment, we assessed nutritional gel (NG) supplementation as a support strategy for mice infected with mouse-adapted Influenza A/Puerto Rico/8/34 (A/PR/8/34; H1N1) virus. We hypothesized that, compared with the standard of care (SOC), supplementation with NG would reduce weight loss and increase survival in mice infected with IAV without impacting the initial immune response to infection. To assess the effects of NG, male and female C57BL/6J mice were infected with IAV at low, intermediate, or high doses. When compared with SOC, mice given NG showed a significant decrease in the maximal percent weight loss at all viral doses in males and at the intermediate dose for females. Mice supplemented with NG had no deaths for either sex at the intermediate dose and a significant increase in survival in males at the high viral dose. Supplementation with NG did not alter the viral titer or the pulmonary recruitment of immune cells as measured by cell counts and flow cytometry of cells recovered in bronchoalveolar lavage (BAL) fluid in either sex. However, mice given NG had a significant reduction in IL6 and TNFα in BAL fluid and no significant differences in CCL2, IL4, IL10, CXCL1, CXCL2, and VEGF. The results of this study show that as compared with infected SOC mice, infected mice supplemented with NG have reduced weight loss and increased survival, with males showing a greater benefit. These results suggest that NG should be considered as a support strategy and indicate that sex is an important biologic variable in mice infected with IAV.

CFTR Modulator Therapy Enhances Peripheral Blood Monocyte Contributions to Immune Responses in People With Cystic Fibrosis.

BACKGROUND: CFTR modulators decrease some etiologies of CF airway inflammation; however, data indicate that non-resolving airway infection and inflammation persist in individuals with CF and chronic bacterial infections. Thus, identification of therapies that diminish airway inflammation without allowing unrestrained bacterial growth remains a critical research goal. Novel strategies for combatting deleterious airway inflammation in the CFTR modulator era require better understanding of cellular contributions to chronic CF airway disease, and how inflammatory cells change after initiation of CFTR modulator therapy. Peripheral blood monocytes, which traffic to the CF airway, can develop both pro-inflammatory and inflammation-resolving phenotypes, represent intriguing cellular targets for focused therapies. This therapeutic approach, however, requires a more detailed knowledge of CF monocyte cellular programming and phenotypes. MATERIAL AND METHODS: In order to characterize the inflammatory phenotype of CF monocytes, and how these cells change after initiation of CFTR modulator therapy, we studied adults (n=10) with CF, chronic airway infections, and the CFTR-R117H mutations before and 7 days after initiation of ivacaftor. Transcriptomes of freshly isolated blood monocytes were interrogated by RNA-sequencing (RNA-seq) followed by pathway-based analyses. Plasma concentrations of cytokines and chemokines were evaluated by multiplex ELISA. RESULTS: RNAseq identified approximately 50 monocyte genes for which basal expression was significantly changed in all 10 subjects after 7 days of ivacaftor. Of these, the majority were increased in expression post ivacaftor, including many genes traditionally associated with enhanced inflammation and immune responses. Pathway analyses confirmed that transcriptional programs were overwhelmingly up-regulated in monocytes after 7 days of ivacaftor, including biological modules associated with immunity, cell cycle, oxidative phosphorylation, and the unfolded protein response. Ivacaftor increased plasma concentrations of CXCL2, a neutrophil chemokine secreted by monocytes and macrophages, and CCL2, a monocyte chemokine. CONCLUSIONS: Our results demonstrate that ivacaftor causes acute changes in blood monocyte transcriptional profiles and plasma chemokines, and suggest that increased monocyte inflammatory signals and changes in myeloid cell trafficking may contribute to changes in airway inflammation in people taking CFTR modulators. To our knowledge, this is the first report investigating the transcriptomic response of circulating blood monocytes in CF subjects treated with a CFTR modulator.

Excitation Emission Matrix Fluorescence Spectroscopy for Combustion Generated Particulate Matter Source Identification.

The inhalation of particulate matter (PM) is a significant health risk associated with reduced life expectancy due to increased cardio-pulmonary disease and exacerbation of respiratory diseases such as asthma and pneumonia. PM originates from natural and anthropogenic sources including combustion engines, cigarettes, agricultural burning, and forest fires. Identifying the source of PM can inform effective mitigation strategies and policies, but this is difficult to do using current techniques. Here we present a method for identifying PM source using excitation emission matrix (EEM) fluorescence spectroscopy and a machine learning algorithm. We collected combustion generated PM2.5 from wood burning, diesel exhaust, and cigarettes using filters. Filters were weighted to determine mass concentration followed by extraction into cyclohexane and analysis by EEM fluorescence spectroscopy. Spectra obtained from each source served as training data for a convolutional neural network (CNN) used for source identification in mixed samples. This method can predict the presence or absence of the three laboratory sources with an overall accuracy of 89% when the threshold for classifying a source as present is 1.1 µg/m3 in air over a 24-hour sampling time. The limit of detection for cigarette, diesel and wood are 0.7, 2.6, 0.9 µg/m3, respectively, in air assuming a 24-hour sampling time at an air sampling rate of 1.8 liters per minute. We applied the CNN algorithm developed using the laboratory training data to a small set of field samples and found the algorithm was effective in some cases but would require a training data set containing more samples to be more broadly applicable.

Ivacaftor decreases monocyte sensitivity to interferon-γ in people with cystic fibrosis.

This study demonstrates that initiation of the CFTR modulator ivacaftor in people with cystic fibrosis and susceptible CFTR mutations causes an acute reduction in blood monocyte sensitivity to the key proinflammatory cytokine IFN-γ http://bit.ly/2TeI6LG.

MEK1 regulates pulmonary macrophage inflammatory responses and resolution of acute lung injury.

The MEK1/2-ERK1/2 pathway has been implicated in regulating the inflammatory response to lung injury and infection, and pharmacologic MEK1/2 inhibitor compounds are reported to reduce detrimental inflammation in multiple animal models of disease, in part through modulation of leukocyte responses. However, the specific contribution of myeloid MEK1 in regulating acute lung injury (ALI) and its resolution remain unknown. Here, the role of myeloid Mek1 was investigated in a murine model of LPS-induced ALI (LPS-ALI) by genetic deletion using the Cre-floxed system (LysMCre × Mekfl), and human alveolar macrophages from healthy volunteers and patients with acute respiratory distress syndrome (ARDS) were obtained to assess activation of the MEK1/2-ERK1/2 pathway. Myeloid Mek1 deletion results in a failure to resolve LPS-ALI, and alveolar macrophages lacking MEK1 had increased activation of MEK2 and the downstream target ERK1/2 on day 4 of LPS-ALI. The clinical significance of these findings is supported by increased activation of the MEK1/2-ERK1/2 pathway in alveolar macrophages from patients with ARDS compared with alveolar macrophages from healthy volunteers. This study reveals a critical role for myeloid MEK1 in promoting resolution of LPS-ALI and controlling the duration of macrophage proinflammatory responses.

Deletion of LysM in LysMCre Recombinase Homozygous Mice is Non-contributory in LPS-Induced Acute Lung Injury.

Lysozyme is an important component of the innate immune system and has roles in peptidoglycan cleavage of gram-positive organisms. Myeloid cells highly express the isoform, lysozyme M, and its promoter has been used to direct Cre recombinase expression to target deletion of floxed genes in myeloid cells. However, generation of the LysMCre mouse effectively disrupts the LysM gene, and mice homozygous for the Cre allele lack the LysM gene product. To test the contribution of LysM in sterile acute lung injury, we generated LysMCre mice homozygous for the Cre allele (+/+) or wild-type allele (-/-). These mice were challenged with LPS delivered via oropharygneal aspiration. Mice were monitored and weighed daily, and BAL cell counts, differential, protein, and cytokine levels were assessed at days 2 and 4. LysMCre+/+ and LysMCre-/- had similar weight loss and recovery, and similar inflammatory responses to LPS at days 2 and 4. These findings indicate that loss of LysM and expression of Cre recombinase are non-contributory in sterile acute lung injury.

Alveolar Macrophage Transcriptional Programs Are Associated with Outcomes in Acute Respiratory Distress Syndrome.

Rationale: Serial measurements of alveolar macrophage (AM) transcriptional changes in patients with acute respiratory distress syndrome (ARDS) could identify cell-specific biological programs that are associated with clinical outcomes.Objectives: To determine whether AM transcriptional programs are associated with prolonged mechanical ventilation and 28-day mortality in individuals with ARDS.Methods: We performed genome-wide transcriptional profiling of AMs purified from BAL fluid collected from 35 subjects with ARDS. Cells were obtained at baseline (Day 1), Day 4, and Day 8 after ARDS onset (N = 68 total samples). We identified biological pathways that were enriched at each time point in subjects alive and extubated within 28 days after ARDS onset (alive/extubatedDay28) versus those dead or persistently supported on mechanical ventilation at Day 28 (dead/intubatedDay28).Measurements and Main Results: "M1-like" (classically activated) and proinflammatory gene sets such as IL-6/JAK/STAT5 (Janus kinase/signal transducer and activator of transcription 5) signaling were significantly enriched in AMs isolated on Day 1 in alive/extubatedDay28 versus dead/intubatedDay28 subjects. In contrast, by Day 8, many of these same proinflammatory gene sets were enriched in AMs collected from dead/intubatedDay28 compared with alive/extubatedDay28 subjects. Serially sampled alive/extubatedDay28 subjects were characterized by an AM temporal expression pattern of Day 1 enrichment of innate immune programs followed by prompt downregulation on Days 4 and 8. Dead/intubatedDay28 subjects exhibited an opposite pattern, characterized by progressive upregulation of proinflammatory programs over the course of ARDS. The relationship between AM expression profiles and 28-day clinical status was distinct in subjects with direct (pulmonary) versus indirect (extrapulmonary) ARDS.Conclusions: Clinical outcomes in ARDS are associated with highly distinct AM transcriptional programs.

A Flow Cytometric Method for Isolating Cystic Fibrosis Airway Macrophages from Expectorated Sputum.

Research to understand the contribution of macrophages to nonresolving airway inflammation in cystic fibrosis (CF) and other chronic suppurative airways diseases has been hindered by a lack of methods for isolating and studying these cells. With the development of technologies that can characterize small numbers of cells or individual cells, there is an even greater need for methodologies to isolate rare cells in heterogeneous specimens. Here, we describe a method that overcomes the technical obstacles imposed by sputum debris and apoptotic cells, and allows isolation of pure populations of macrophages from CF sputum. In addition to enhancing our ability to study human CF airway macrophages, this protocol can be adapted to study cells in sputum from other chronic suppurative lung diseases (e.g., chronic obstructive pulmonary disease) and used for isolation of individual cells for single cell analyses.

Transcriptional and functional diversity of human macrophage repolarization.

BACKGROUND: Macrophage plasticity allows cells to adopt different phenotypes, a property with important implications in disorders such as cystic fibrosis (CF) and asthma. OBJECTIVE: We sought to examine the transcriptional and functional significance of macrophage repolarization from an M1 to an M2 phenotype and assess the role of a common human genetic disorder (CF) and a prototypical allergic disease (asthma) in this transformation. METHODS: Monocyte-derived macrophages were collected from healthy subjects and patients with CF and polarized to an M2 state by using IL-4, IL-10, glucocorticoids, apoptotic PMNs, or azithromycin. We performed transcriptional profiling and pathway analysis for each stimulus. We assessed the ability of M2-repolarized macrophages to respond to LPS rechallenge and clear apoptotic neutrophils and used murine models to determine conserved functional responses to IL-4 and IL-10. We investigated whether M2 signatures were associated with alveolar macrophage phenotypes in asthmatic patients. RESULTS: We found that macrophages exhibit highly diverse responses to distinct M2-polarizing stimuli. Specifically, IL-10 activated proinflammatory pathways and abrogated LPS tolerance, allowing rapid restoration of LPS responsiveness. In contrast, IL-4 enhanced LPS tolerance, dampening proinflammatory responses after repeat LPS challenge. A common theme observed across all M2 stimuli was suppression of interferon-associated pathways. We found that CF macrophages had intact reparative and transcriptional responses, suggesting that macrophage contributions to CF-related lung disease are primarily shaped by their environment. Finally, we leveraged in vitro-derived signatures to show that allergen provocation induces distinct M2 state transcriptional patterns in alveolar macrophages. CONCLUSION: Our findings highlight the diversity of macrophage polarization, attribute functional consequences to different M2 stimuli, and provide a framework to phenotype macrophages in disease states.

Matrix metalloproteinase 28 is regulated by TRIF- and type I IFN-dependent signaling in macrophages.

Matrix metalloproteinases (MMPs) are transcriptionally regulated proteases that have multiple roles in modifying the extracellular matrix (ECM) and inflammatory response. Our previous work identified Mmp28 as a key regulator of inflammation and macrophage polarization during experimental models of pulmonary infection, fibrosis, and chronic smoke exposure. However, the signaling pathways responsible for regulation of macrophage Mmp28 expression remain undefined. This study utilized murine macrophages obtained from wild type, Tlr2-/-, Tlr4-/-, MyD88-/-, Ticam1 Lps2 ( Trifmutant), and Ifnar1-/- mice to test the hypothesis that macrophage Mmp28 expression was dependent on TRIF and type I IFN. Our results support the hypothesis, demonstrating that increased macrophage Mmp28 expression was dependent on type I IFN after LPS and poly(I:C) stimulation. To gain further insight into the function of MMP28, we explored the inflammatory response of macrophages derived from wild type or Mmp28-/- mice to stimulation with poly(I:C). Our data support a role for MMP28 in regulating the macrophage inflammatory response to poly(I:C) because expression of Ccl2, Ccl4, Cxcl10, and Il6 were increased in Mmp28-/- macrophages. Together, these data support a model in which macrophages integrate TRIF- and type I IFN-dependent signaling to coordinate regulation of proteins with the capacity to modify the ECM.