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.

Understanding and addressing the needs of people with cystic fibrosis in the era of CFTR modulator therapy.

Cystic fibrosis is a multiorgan disease caused by impaired function of the cystic fibrosis transmembrane conductance regulator (CFTR). Since the introduction of the CFTR modulator combination elexacaftor-tezacaftor-ivacaftor (ETI), which acts directly on mutant CFTR to enhance its activity, most people with cystic fibrosis (pwCF) have seen pronounced reductions in symptoms, and studies project marked increases in life expectancy for pwCF who are eligible for ETI. However, modulator therapy has not cured cystic fibrosis and the success of CFTR modulators has resulted in immediate questions about the new state of cystic fibrosis disease and clinical challenges in the care of pwCF. In this Series paper, we summarise key questions about cystic fibrosis disease in the era of modulator therapy, highlighting state-of-the-art research and clinical practices, knowledge gaps, new challenges faced by pwCF and the potential for future health-care challenges, and the pressing need for additional therapies to treat the underlying genetic or molecular causes of cystic fibrosis.

Dectin-1-Independent Macrophage Phagocytosis of Mycobacterium abscessus.

Mycobacterium abscessus, a species of nontuberculous mycobacteria (NTM), is an opportunistic pathogen that is readily cleared by healthy lungs but can cause pulmonary infections in people with chronic airway diseases. Although knowledge pertaining to molecular mechanisms of host defense against NTM is increasing, macrophage receptors that recognize M. abscessus remain poorly defined. Dectin-1, a C-type lectin receptor identified as a fungal receptor, has been shown to be a pathogen recognition receptor (PRR) for both M. tuberculosis and NTM. To better understand the role of Dectin-1 in host defense against M. abscessus, we tested whether blocking Dectin-1 impaired the uptake of M. abscessus by human macrophages, and we compared M. abscessus pulmonary infection in Dectin-1-deficient and wild-type mice. Blocking antibody for Dectin-1 did not reduce macrophage phagocytosis of M. abscessus, but did reduce the ingestion of the fungal antigen zymosan. Laminarin, a glucan that blocks Dectin-1 and other PRRs, caused decreased phagocytosis of both M. abscessus and zymosan. Dectin-1-/- mice exhibited no defects in the control of M. abscessus infection, and no differences were detected in immune cell populations between wild type and Dectin-1-/- mice. These data demonstrate that murine defense against M. abscessus pulmonary infection, as well as ingestion of M. abscessus by human macrophages, can occur independent of Dectin-1. Thus, additional PRR(s) recognized by laminarin participate in macrophage phagocytosis of M. abscessus.

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.

Culture independent markers of nontuberculous mycobacterial (NTM) lung infection and disease in the cystic fibrosis airway.

Nontuberculous mycobacteria (NTM) are opportunistic pathogens that affect a relatively small but significant portion of the people with cystic fibrosis (CF), and may cause increased morbidity and mortality in this population. Cultures from the airway are the only test currently in clinical use for detecting NTM. Culture techniques used in clinical laboratories are insensitive and poorly suited for population screening or to follow progression of disease or treatment response. The lack of sensitive and quantitative markers of NTM in the airway impedes patient care and clinical trial design, and has limited our understanding of patterns of acquisition, latency and pathogenesis of disease. Culture-independent markers of NTM infection have the potential to overcome many of the limitations of standard NTM cultures, especially the very slow growth, inability to quantitate bacterial burden, and low sensitivity due to required decontamination procedures. A range of markers have been identified in sputum, saliva, breath, blood, urine, as well as radiographic studies. Proposed markers to detect presence of NTM or transition to NTM disease include bacterial cell wall products and DNA, as well as markers of host immune response such as immunoglobulins and the gene expression of circulating leukocytes. In all cases the sensitivity of culture-independent markers is greater than standard cultures; however, most do not discriminate between various NTM species. Thus, each marker may be best suited for a specific clinical application, or combined with other markers and traditional cultures to improve diagnosis and monitoring of treatment response.

The Impact of Highly Effective Modulator Therapy on Cystic Fibrosis Microbiology and Inflammation.

Highly effective cystic fibrosis (CF) transmembrane conductance regulator (CFTR) modulator therapy (HEMT) corrects the underlying molecular defect causing CF disease. HEMT decreases symptom burden and improves clinical metrics and quality of life for most people with CF (PwCF) and eligible cftr mutations. Improvements in measures of pulmonary health suggest that restoration of function of defective CFTR anion channels by HEMT not only enhances airway mucociliary clearance, but also reduces chronic pulmonary infection and inflammation. This article reviews the evidence for how HEMT influences the dynamic and interdependent processes of infection and inflammation in the CF airway, and what questions remain unanswered.

Acute Pulmonary Exacerbation Phenotypes in Patients with Cystic Fibrosis.

Rationale: The etiology of cystic fibrosis (CF) pulmonary exacerbations (PEx) is likely multifactorial with viral, bacterial, and non-infectious pathways contributing. Objectives: To determine whether viral infection status and CRP (C-reactive protein) can classify subphenotypes of PEx that differ in outcomes and biomarker profiles. Methods: Patients were recruited at time of admission for a PEx. Nasal swabs and sputum samples were collected and processed using the respiratory panel of the FilmArray multiplex polymerase chain reaction (PCR). Serum and plasma biomarkers were measured. PEx were classified using serum CRP and viral PCR: "pauci-inflammatory" if CRP < 5 mg/L, "non-viral with systemic inflammation" if CRP ⩾ 5 mg/L and no viral infection detected by PCR and "viral with systemic inflammation" if CRP ⩾ 5 mg/L and viral infection detected by PCR. Results: Discovery cohort (n = 59) subphenotype frequencies were 1) pauci-inflammatory (37%); 2) non-viral with systemic inflammation (41%); and 3) viral with systemic inflammation (22%). Immunoglobulin G, immunoglobulin M, interleukin-10, interleukin-13, serum calprotectin, and CRP levels differed across phenotypes. Reduction from baseline in forced expiratory volume in 1 second as percent predicted (FEV1pp) at onset of exacerbation differed between non-viral with systemic inflammation and viral with systemic inflammation (-6.73 ± 1.78 vs. -13.5 ± 2.32%; P = 0.025). Non-viral with systemic inflammation PEx had a trend toward longer duration of intravenous antibiotics versus pauci-inflammation (18.1 ± 1.17 vs. 14.8 ± 1.19 days, P = 0.057). There were no differences in percent with lung function recovery to <10% of baseline FEV1pp. Similar results were seen in local and external validation cohorts comparing a pauci-inflammatory to viral/non-viral inflammatory exacerbation phenotypes. Conclusions: Subphenotypes of CF PEx exist with differences in biomarker profile, clinical presentation, and outcomes.

Host and pathogen response to bacteriophage engineered against Mycobacterium abscessus lung infection.

Two mycobacteriophages were administered intravenously to a male with treatment-refractory Mycobacterium abscessus pulmonary infection and severe cystic fibrosis lung disease. The phages were engineered to enhance their capacity to lyse M. abscessus and were selected specifically as the most effective against the subject's bacterial isolate. In the setting of compassionate use, the evidence of phage-induced lysis was observed using molecular and metabolic assays combined with clinical assessments. M. abscessus isolates pre and post-phage treatment demonstrated genetic stability, with a general decline in diversity and no increased resistance to phage or antibiotics. The anti-phage neutralizing antibody titers to one phage increased with time but did not prevent clinical improvement throughout the course of treatment. The subject received lung transplantation on day 379, and systematic culturing of the explanted lung did not detect M. abscessus. This study describes the course and associated markers of a successful phage treatment of M. abscessus in advanced lung disease.

Pseudomonas aeruginosa aggregates in cystic fibrosis sputum produce exopolysaccharides that likely impede current therapies.

In cystic fibrosis (CF) airways, Pseudomonas aeruginosa forms cellular aggregates called biofilms that are thought to contribute to chronic infection. To form aggregates, P. aeruginosa can use different mechanisms, each with its own pathogenic implications. However, how they form in vivo is controversial and unclear. One mechanism involves a bacterially produced extracellular matrix that holds the aggregates together. Pel and Psl exopolysaccharides are structural and protective components of this matrix. We develop an immunohistochemical method to visualize Pel and Psl in CF sputum. We demonstrate that both exopolysaccharides are expressed in the CF airways and that the morphology of aggregates is consistent with an exopolysaccharide-dependent aggregation mechanism. We reason that the cationic exopolysaccharide Pel may interact with some of the abundant anionic host polymers in sputum. We show that Pel binds extracellular DNA (eDNA) and that this interaction likely impacts current therapies by increasing antimicrobial tolerance and protecting eDNA from digestion.

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.

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.

Strong toll-like receptor responses in cystic fibrosis patients are associated with higher lung function.

BACKGROUND: Cystic fibrosis (CF) airways disease varies widely among patients with identical cystic fibrosis transmembrane conductance regulator (CFTR) genotypes. Robust airway inflammation is thought to be deleterious in CF; inter-individual variation in Toll-like receptor (TLR)-mediated innate immune inflammatory responses (TMIIR) might account for a portion of the phenotypic variation. We tested if TMIIR in people with CF are different than those of healthy controls, and whether higher TMIIR in people with CF are associated with reduced lung function. METHODS: We cultured whole blood from clinically stable subjects with CF (n = 76) and healthy controls (n = 45) with TLR agonists, and measured cytokine production and expression of TLR-associated genes. We tested for differences in TLR-stimulated cytokine levels between subjects with CF and healthy subjects, and for associations between cytokine and gene expression levels with baseline lung function (forced expiratory volume in one second percent predicted (FEV1%)) and decline in FEV1% over time. RESULTS: TMIIR in blood from subjects with CF were lower than in healthy controls. Expression of TLR regulators SARM1, TOLLIP, and AKT1 were downregulated in CF. In subjects with CF we found that lower TLR4-agonist-induced IL-8 was associated with lower FEV1% at enrollment (p<0.001) and with greater five year FEV1% decline (p<0.001). CONCLUSIONS: TMIIR were lower in people with CF relative to healthy controls; however, unexpectedly, greater whole blood TMIIR were positively associated with lung function in people with CF. These findings suggest a complex interaction between inflammation and disease in people with CF.

In Vivo Proteome of Pseudomonas aeruginosa in Airways of Cystic Fibrosis Patients.

Chronic airway infection with P. aeruginosa (PA) is a hallmark of cystic fibrosis (CF) disease. The mechanisms producing PA persistence in CF therapies remain poorly understood. To gain insight on PA physiology in patient airways and better understand how in vivo bacterial functioning differs from in vitro conditions, we investigated the in vivo proteomes of PA in 35 sputum samples from 11 CF patients. We developed a novel bacterial-enrichment method that relies on differential centrifugation and detergent treatment to enrich for bacteria to improve identification of PA proteome with CF sputum samples. Using two nonredundant peptides as a cutoff, a total of 1304 PA proteins were identified directly from CF sputum samples. The in vivo PA proteomes were compared with the proteomes of ex vivo-grown PA populations from the same patient sample. Label-free quantitation and proteome comparison revealed the in vivo up-regulation of siderophore TonB-dependent receptors, remodeling in central carbon metabolism including glyoxylate cycle and lactate utilization, and alginate overproduction. Knowledge of these in vivo proteome differences or others derived using the presented methodology could lead to future treatment strategies aimed at altering PA physiology in vivo to compromise infectivity or improve antibiotic efficacy.

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.

Restoring Cystic Fibrosis Transmembrane Conductance Regulator Function Reduces Airway Bacteria and Inflammation in People with Cystic Fibrosis and Chronic Lung Infections.

RATIONALE: Previous work indicates that ivacaftor improves cystic fibrosis transmembrane conductance regulator (CFTR) activity and lung function in people with cystic fibrosis and G551D-CFTR mutations but does not reduce density of bacteria or markers of inflammation in the airway. These findings raise the possibility that infection and inflammation may progress independently of CFTR activity once cystic fibrosis lung disease is established. OBJECTIVES: To better understand the relationship between CFTR activity, airway microbiology and inflammation, and lung function in subjects with cystic fibrosis and chronic airway infections. METHODS: We studied 12 subjects with G551D-CFTR mutations and chronic airway infections before and after ivacaftor. We measured lung function, sputum bacterial content, and inflammation, and obtained chest computed tomography scans. MEASUREMENTS AND MAIN RESULTS: Ivacaftor produced rapid decreases in sputum Pseudomonas aeruginosa density that began within 48 hours and continued in the first year of treatment. However, no subject eradicated their infecting P. aeruginosa strain, and after the first year P. aeruginosa densities rebounded. Sputum total bacterial concentrations also decreased, but less than P. aeruginosa. Sputum inflammatory measures decreased significantly in the first week of treatment and continued to decline over 2 years. Computed tomography scans obtained before and 1 year after ivacaftor treatment revealed that ivacaftor decreased airway mucous plugging. CONCLUSIONS: Ivacaftor caused marked reductions in sputum P. aeruginosa density and airway inflammation and produced modest improvements in radiographic lung disease in subjects with G551D-CFTR mutations. However, P. aeruginosa airway infection persisted. Thus, measures that control infection may be required to realize the full benefits of CFTR-targeting treatments.

Filamentous Bacteriophage Produced by Pseudomonas aeruginosa Alters the Inflammatory Response and Promotes Noninvasive Infection In Vivo.

Pseudomonas aeruginosa is an important opportunistic human pathogen that lives in biofilm-like cell aggregates at sites of chronic infection, such as those that occur in the lungs of patients with cystic fibrosis and nonhealing ulcers. During growth in a biofilm, P. aeruginosa dramatically increases the production of filamentous Pf bacteriophage (Pf phage). Previous work indicated that when in vivo Pf phage production was inhibited, P. aeruginosa was less virulent. However, it is not clear how the production of abundant quantities of Pf phage similar to those produced by biofilms under in vitro conditions affects pathogenesis. Here, using a murine pneumonia model, we show that the production of biofilm-relevant amounts of Pf phage prevents the dissemination of P. aeruginosa from the lung. Furthermore, filamentous phage promoted bacterial adhesion to mucin and inhibited bacterial invasion of airway epithelial cultures, suggesting that Pf phage traps P. aeruginosa within the lung. The in vivo production of Pf phage was also associated with reduced lung injury, reduced neutrophil recruitment, and lower cytokine levels. Additionally, when producing Pf phage, P. aeruginosa was less prone to phagocytosis by macrophages than bacteria not producing Pf phage. Collectively, these data suggest that filamentous Pf phage alters the progression of the inflammatory response and promotes phenotypes typically associated with chronic infection.

Acute administration of ivacaftor to people with cystic fibrosis and a G551D-CFTR mutation reveals smooth muscle abnormalities.

BACKGROUND: Airflow obstruction is common in cystic fibrosis (CF), yet the underlying pathogenesis remains incompletely understood. People with CF often exhibit airway hyperresponsiveness, CF transmembrane conductance regulator (CFTR) is present in airway smooth muscle (ASM), and ASM from newborn CF pigs has increased contractile tone, suggesting that loss of CFTR causes a primary defect in ASM function. We hypothesized that restoring CFTR activity would decrease smooth muscle tone in people with CF. METHODS: To increase or potentiate CFTR function, we administered ivacaftor to 12 adults with CF with the G551D-CFTR mutation; ivacaftor stimulates G551D-CFTR function. We studied people before and immediately after initiation of ivacaftor (48 hours) to minimize secondary consequences of CFTR restoration. We tested smooth muscle function by investigating spirometry, airway distensibility, and vascular tone. RESULTS: Ivacaftor rapidly restored CFTR function, indicated by reduced sweat chloride concentration. Airflow obstruction and air trapping also improved. Airway distensibility increased in airways less than 4.5 mm but not in larger-sized airways. To assess smooth muscle function in a tissue outside the lung, we measured vascular pulse wave velocity (PWV) and augmentation index, which both decreased following CFTR potentiation. Finally, change in distensibility of <4.5-mm airways correlated with changes in PWV. CONCLUSIONS: Acute CFTR potentiation provided a unique opportunity to investigate CFTR-dependent mechanisms of CF pathogenesis. The rapid effects of ivacaftor on airway distensibility and vascular tone suggest that CFTR dysfunction may directly cause increased smooth muscle tone in people with CF and that ivacaftor may relax smooth muscle. FUNDING: This work was funded in part from an unrestricted grant from the Vertex Investigator-Initiated Studies Program.