In Cystic Fibrosis (CF), the airways are often colonized by opportunistic fungi. The most frequently detected mold is Aspergillus fumigatus (Af). Af diseases are associated with significant morbidity and mortality. The most common clinical picture caused by Af is allergic bronchopulmonary aspergillosis (ABPA), triggered by an immunological reaction against Af. Af bronchitis and invasive aspergillosis rarely occur in CF as a result of spore colonization and germination. Since pulmonary mycoses and exacerbations by other pathogens overlap in clinical, radiological, and immunological characteristics, diagnosis still remains a challenge. The search for reliable, widely available biomarkers for Af diseases is therefore still an important task today.

Pseudomonas aeruginosa is the archetypal cystic fibrosis (CF) pathogen. However, the clinical course experienced by infected individuals varies markedly. Understanding these differences is imperative if further improvements in outcomes are to be achieved. Multiple studies have found that patients infected with epidemic P. aeruginosa (ePA) strains may have a worse clinical prognosis than those infected with unique, non-clonal strains. Additionally, the traditionally uncultured CF lung bacterial community (i.e., CF microbiome) may further influence the outcome. We sought to identify if these two important variables, not identified through routine culture, associate and together may contribute to disease pathogenesis. Patients were classified as being infected with Prairie Epidemic ePA (PES) or a non-clonal strain, unique PA strains (uPA), through a retrospective assessment of a comprehensive strain biobank using a combination of PFGE and PES-specific PCR. Patients were matched to age, sex, time-period controls and sputum samples from equivalent time periods were identified from a sputum biobank. Bacterial 16S rRNA gene profiling and Pseudomonas qPCR was used to characterize the respiratory microbiome. We identified 31 patients infected with PES and matched them with uPA controls. Patients infected with PES at baseline have lower microbial diversity (P = 0.02) and higher P. aeruginosa relative abundance (P < 0.005). Microbial community structure was found to cluster by PA strain type, although it was not the main determinant of community structure as additional factors were also found to be drivers of CF community structure. Communities from PES infected individuals were enriched with Pseudomonas, Streptococcus and Prevotella OTUs. The disproportionate disease experienced by ePA infected CF patients may be mediated through a combination of pathogen-pathogen factors as opposed to strictly enhanced virulence of infecting P. aeruginosa strains.

Hallmarks of cystic fibrosis (CF) are increased viscosity of mucus and impaired mucociliary clearance within the airways due to mutations of the cystic fibrosis conductance regulator gene. This facilitates the colonization of the lung by microbial pathogens and the concomitant establishment of chronic infections leading to tissue damage, reduced lung function, and decreased life expectancy. Although the interplay between key CF pathogens plays a major role during disease progression, the pathophysiology of the microbial community in CF lungs remains poorly understood. Particular challenges in the analysis of the microbial population present in CF sputum is (I) the inhomogeneous, viscous, and slimy consistence of CF sputum, and (II) the high number of human proteins masking comparably low abundant microbial proteins. To address these challenges, we used 21 CF sputum samples to develop a reliable, reproducible and widely applicable protocol for sputum processing, microbial enrichment, cell disruption, protein extraction and subsequent metaproteomic analyses. As a proof of concept, we selected three sputum samples for detailed metaproteome analyses and complemented and validated metaproteome data by 16S sequencing, metabolomic as well as microscopic analyses. Applying our protocol, the number of bacterial proteins/protein groups increased from 199-425 to 392-868 in enriched samples compared to nonenriched controls. These early microbial metaproteome data suggest that the arginine deiminase pathway and multiple proteases and peptidases identified from various bacterial genera could so far be underappreciated in their contribution to the CF pathophysiology. By providing a standardized and effective protocol for sputum processing and microbial enrichment, our study represents an important basis for future studies investigating the physiology of microbial pathogens in CF in vivo - an important prerequisite for the development of novel antimicrobial therapies to combat chronic recurrent airway infection in CF.

Chronic Pseudomonas aeruginosa infections play an important role in the progress of lung disease in patients suffering from cystic fibrosis (CF). Recent studies indicate that polymicrobial microbiome profiles in the airway are associated with less inflammation. Thus, the hypothesis was raised that certain commensal bacteria might protect the host from inflammation. We therefore performed a screening study with commensals isolated from CF airway microbiome samples to identify potential beneficial commensals. We isolated more than 80 aerobic or facultative anaerobic commensal strains, including strains from genera Streptococcus, Neisseria, Actinomyces, Corynebacterium, Dermabacter, Micrococcus and Rothia. Through a screening experiment of co-infection in human epithelial cell lines, we identified multiple commensal strains, especially strains belonging to Streptococcus mitis, that reduced P. aeruginosa triggered inflammatory responses. The results were confirmed by co-infection experiments in ex-vivo precision cut lung slices (PCLS) from mice. The underlying mechanisms of the complex host-pathogen-commensal crosstalk were investigated from both the host and the bacterial sides with a focus on S. mitis. Transcriptome changes in the host in response to co-infection and mono-infection were evaluated, and the results indicated that several signalling pathways mediating inflammatory responses were downregulated by co-infection with S. mitis and P. aeruginosa compared to P. aeruginosa mono-infection, such as neutrophil extracellular trap formation. The genomic differences among S. mitis strains with and without protective effects were investigated by whole genome sequencing, revealing genes only present in the S. mitis strains showing protective effects. In summary, through both in vitro and ex vivo studies, we could identify a variety of commensal strains that may reduce host inflammatory responses induced by P. aeruginosa infection. These findings support the hypothesis that CF airway commensals may protect the host from inflammation.

The detection and diagnosis of the opportunistic fungi Scedosporium spp. and Lomentospora prolificans still relies mainly on low-sensitive culture-based methods. This fact is especially worrying in Cystic Fibrosis (CF) patients in whom these fungal species are frequently isolated and may increase the risk of suffering from an infection or other health problems. Therefore, with the purpose of developing a serologic detection method for Scedosporium/Lomentospora, four different Scedosporium boydii protein extracts (whole cell protein extract, secretome, total cell surface and conidial surface associated proteins) were studied by ELISA to select the most useful for IgG detection in sera from CF patients. The four extracts were able to discriminate the Scedosporium/Lomentospora-infected from Aspergillus-infected and non-infected patients. However, the whole cell protein extract was the one selected, as it was the one with the highest output in terms of protein concentration per ml of fungal culture used, and its discriminatory capacity was the best. The ELISA test developed was then assayed with 212 sera from CF patients and it showed to be able to detect Scedosporium spp. and Lomentospora prolificans with very high sensitivity and specificity, 86%-100% and 93%-99%, respectively, depending on the cut-off value chosen (four values were proposed A450nm= 0.5837, A450nm= 0.6042, A450nm= 0.6404, and A450nm= 0.7099). Thus, although more research is needed to reach a standardized method, this ELISA platform offers a rapid, low-cost and easy solution to detect these elusive fungi through minimally invasive sampling, allowing the monitoring of the humoral response to fungal presence.

Microbial communities infiltrate the respiratory tract of cystic fibrosis patients, where chronic colonization and infection lead to clinical decline. This report aims to provide an overview of the diversity of bacterial and fungal species from the airway secretion of three young CF patients with severe pulmonary disease. The bacterial and fungal microbiomes were investigated by culture isolation, metataxonomics, and metagenomics shotgun. Virulence factors and antibiotic resistance genes were also explored. A. fumigatus was isolated from cultures and identified in high incidence from patient sputum samples. Candida albicans, Penicillium sp., Hanseniaspora sp., Torulaspora delbrueckii, and Talaromyces amestolkiae were isolated sporadically. Metataxonomics and metagenomics detected fungal reads (Saccharomyces cerevisiae, A. fumigatus, and Schizophyllum sp.) in one sputum sample. The main pathogenic bacteria identified were Staphylococcus aureus, Pseudomonas aeruginosa, Burkholderia cepacia complex, and Achromobacter xylosoxidans. The canonical core CF microbiome is composed of species from the genera Streptococcus, Neisseria, Rothia, Prevotella, and Haemophilus. Thus, the airways of the three young CF patients presented dominant bacterial genera and interindividual variability in microbial community composition and diversity. Additionally, a wide diversity of virulence factors and antibiotic resistance genes were identified in the CF lung microbiomes, which may be linked to the clinical condition of the CF patients. Understanding the microbial community is crucial to improve therapy because it may have the opposite effect, restructuring the pathogenic microbiota. Future studies focusing on the influence of fungi on bacterial diversity and microbial interactions in CF microbiomes will be welcome to fulfill this huge gap of fungal influence on CF physiopathology.

Cystic fibrosis (CF) human and mouse macrophages are defective in their ability to clear bacteria such as Burkholderia cenocepacia. The autophagy process in CF (F508del) macrophages is halted, and the underlying mechanism remains unclear. Furthermore, the role of CFTR in maintaining the acidification of endosomal and lysosomal compartments in CF cells has been a subject of debate. Using 3D reconstruction of z-stack confocal images, we show that CFTR is recruited to LC3-labeled autophagosomes harboring B. cenocepacia. Using several complementary approaches, we report that CF macrophages display defective lysosomal acidification and degradative function for cargos destined to autophagosomes, whereas non-autophagosomal cargos are effectively degraded within acidic compartments. Notably, treatment of CF macrophages with CFTR modulators (tezacaftor/ivacaftor) improved the autophagy flux, lysosomal acidification and function, and bacterial clearance. In addition, CFTR modulators improved CFTR function as demonstrated by patch-clamp. In conclusion, CFTR regulates the acidification of a specific subset of lysosomes that specifically fuse with autophagosomes. Therefore, our study describes a new biological location and function for CFTR in autophago-lysosomes and clarifies the long-standing discrepancies in the field.

Biofilm-producing strains of Pseudomonas aeruginosa are a major cause of morbidity and mortality in cystic fibrosis (CF) patients. In these patients, increased levels of IL-17 as well as of IL-5 and IL-13 along with arginase (Arg)-positive macrophages have been observed in bronchoalveolar lavage fluid. While IL-17 is a strong proinflammatory cytokine associated with host defense against bacterial and fungal infections and is also elevated in several autoimmune diseases, IL-5/IL-13 and Arg1-positive M2 macrophages are part of the anti-inflammatory type 2 (Th2) immunity. To study whether increased IL-5 and IL-13 levels are related to biofilm formation, which is frequently observed in CF patients colonized by P. aeruginosa, we utilized an agarose bead-embedded P. aeruginosa rat model commonly employed in in vivo biofilm studies. We showed that "sterile" agarose bead instillation in rat notably increased lung transcript levels of IL-5 and IL-13 at two post-instillation study-points, day 1 and day 3. Concurrently, increased infiltration of type 2 innate cells such as eosinophils and Arg1 positive M2 activated macrophages (Arg1+CD68+) was also observed both at day 1 and day 3 while the proportion of M1 activated macrophages (iNOS+CD68+) at these time-points decreased. In contrast, P. aeruginosa-loaded beads caused a drastic elevation of proinflammatory Th1 (IFNγ, TNFα, IL-12a) and antibacterial Th17 (IL-17a, IL-17f, IL-22, IL-23a) cytokines along with a high influx of neutrophils and M1 macrophages, while Th2 cytokines (IL-5 and IL-13) drastically declined at day 1 post-infection. Interestingly, at day 3 post-infection, both Th1 and Th17 cytokines sharply declined and corroborated with decreased M1 and increased M2 macrophages. These data suggest that while IL-17 is linked to episodes of acute exacerbations of infection in CF patients, the increased Th2 cytokines and M2 macrophages observed in these patients are largely due to the biofilm matrix. The data presented here has important implications for clinical management of CF patients.

The identification of 16S rDNA biomarkers from respiratory samples to describe the continuum of clinical disease states within persons having cystic fibrosis (CF) has remained elusive. We sought to combine 16S, metagenomics, and metabolomics data to describe multiple transitions between clinical disease states in 14 samples collected over a 12-month period in a single person with CF. We hypothesized that each clinical disease state would have a unique combination of bacterial genera and volatile metabolites as a potential signature that could be utilized as a biomarker of clinical disease state. Taxonomy identified by 16S sequencing corroborated clinical culture results, with the majority of the 109 PCR amplicons belonging to the bacteria grown in clinical cultures (Escherichia coli and Staphylococcus aureus). While alpha diversity measures fluctuated across disease states, no significant trends were present. Principle coordinates analysis showed that treatment samples trended toward a different community composition than baseline and exacerbation samples. This was driven by the phylum Bacteroidetes (less abundant in treatment, log2 fold difference -3.29, p = 0.015) and the genus Stenotrophomonas (more abundant in treatment, log2 fold difference 6.26, p = 0.003). Across all sputum samples, 466 distinct volatile metabolites were identified with total intensity varying across clinical disease state. Baseline and exacerbation samples were rather uniform in chemical composition and similar to one another, while treatment samples were highly variable and differed from the other two disease states. When utilizing a combination of the microbiome and metabolome data, we observed associations between samples dominated Staphylococcus and Escherichia and higher relative abundances of alcohols, while samples dominated by Achromobacter correlated with a metabolomics shift toward more oxidized volatiles. However, the microbiome and metabolome data were not tightly correlated; examining both the metagenomics and metabolomics allows for more context to examine changes across clinical disease states. In our study, combining the sputum microbiome and metabolome data revealed stability in the sputum composition through the first exacerbation and treatment episode, and into the second exacerbation. However, the second treatment ushered in a prolonged period of instability, which after three additional exacerbations and treatments culminated in a new lung microbiome and metabolome.

Pseudomonas aeruginosa is a significant cause of mortality in patients with cystic fibrosis (CF). To explore the interaction of the CF isolate P. aeruginosa PASS1 with the innate immune response, we have used Danio rerio (zebrafish) as an infection model. Confocal laser scanning microscopy (CLSM) enabled visualization of direct interactions between zebrafish macrophages and P. aeruginosa PASS1. Dual RNA-sequencing of host-pathogen was undertaken to profile RNA expression simultaneously in the pathogen and the host during P. aeruginosa infection. Following establishment of infection in zebrafish embryos with PASS1, 3 days post infection (dpi), there were 6739 genes found to be significantly differentially expressed in zebrafish and 176 genes in PASS1. A range of virulence genes were upregulated in PASS1, including genes encoding pyoverdine biosynthesis, flagellin, non-hemolytic phospholipase C, proteases, superoxide dismutase and fimbrial subunits. Additionally, iron and phosphate acquisition genes were upregulated in PASS1 cells in the zebrafish. Transcriptional changes in the host immune response genes highlighted phagocytosis as a key response mechanism to PASS1 infection. Transcriptional regulators of neutrophil and macrophage phagocytosis were upregulated alongside transcriptional regulators governing response to tissue injury, infection, and inflammation. The zebrafish host showed significant downregulation of the ribosomal RNAs and other genes involved in translation, suggesting that protein translation in the host is affected by PASS1 infection.

Bacteria of the genus Achromobacter are environmental germs, with an unknown reservoir. It can become opportunistic pathogens in immunocompromised patients, causing bacteremia, meningitis, pneumonia, or peritonitis. In recent years, Achromobacter xylosoxidans has emerged with increasing incidence in patients with cystic fibrosis (CF). Recent studies showed that A. xylosoxidans is involved in the degradation of the respiratory function of patients with CF. The respiratory ecosystem of patients with CF is colonized by bacterial species that constantly fight for space and access to nutrients. The type VI secretion system (T6SS) empowers this constant bacterial antagonism, and it is used as a virulence factor in several pathogenic bacteria. This study aimed to investigate the prevalence of the T6SS genes in A. xylosoxidans isolated in patients with CF. We also evaluated clinical and molecular characteristics of T6SS-positive A. xylosoxidans strains. We showed that A. xylosoxidans possesses a T6SS gene cluster and that some environmental and clinical isolates assemble a functional T6SS nanomachine. A. xylosoxidans T6SS is used to target competing bacteria, including other CF-specific pathogens. Finally, we demonstrated the importance of the T6SS in the internalization of A. xylosoxidans in lung epithelial cells and that the T6SS protein Hcp is detected in the sputum of patients with CF. Altogether, these results suggest for the first time a role of T6SS in CF-lung colonization by A. xylosoxidans and opens promising perspective to target this virulence determinant as innovative theranostic options for CF management.

Lung infection with the fungus Aspergillus fumigatus (Af) is a common complication in cystic fibrosis (CF) and is associated with loss of pulmonary function. We established a fungal epithelial co-culture model to examine the impact of Af infection on CF bronchial epithelial barrier function using Af strains 10AF and AF293-GFP, and the CFBE41o- cell line homozygous for the F508del mutation with (CF+CFTR) and without (CF) normal CFTR expression. Following exposure of the epithelial surface to Af conidia, formation of germlings (early stages of fungal growth) was detected after 9-12 hours and hyphae (mature fungal growth) after 12-24 hours. During fungal morphogenesis, bronchial epithelial cells showed signs of damage including rounding, and partial detachment after 24 hours. Fluorescently labeled conidia were internalized after 6 hours and more internalized conidia were observed in CF compared to CF+CFTR cells. Infection of the apical surface with 10AF conidia, germlings, or hyphae was performed to determine growth stage-specific effects on tight junction protein zona occludens protein 1 (ZO-1) expression and transepithelial electrical resistance (TER). In response to infection with conidia or germlings, epithelial barrier function degraded time-dependently (based on ZO-1 immunofluorescence and TER) with a delayed onset in CF+CFTR cell monolayers and required viable fungi and apical application. Infection with hyphae caused an earlier onset and faster rate of decline in TER compared to conidia and germlings. Gliotoxin, a major Af virulence factor, caused a rapid decline in TER and induced a transient chloride secretory response in CF+CFTR but not CF cells. Our findings suggest growth and internalization of Af result in deleterious effects on bronchial epithelial barrier function that occurred more rapidly in the absence of CFTR. Bronchial epithelial barrier breakdown was time-dependent and morphotype-specific and mimicked by acute administration of gliotoxin. Our study also suggests a protective role for CFTR by turning on CFTR-dependent chloride transport in response to gliotoxin, a mechanism that will support mucociliary clearance, and could delay the loss of epithelial integrity during fungal development in vivo.

Scedosporium and Lomentospora species are filamentous fungi that cause a wide range of infections in humans. They are usually found in the lungs of cystic fibrosis (CF) patients and are the second most frequent fungal genus after Aspergillus species. Several studies have been recently performed in order to understand how fungi and bacteria interact in CF lungs, since both can be isolated simultaneously from patients. In this context, many bacterial molecules were shown to inhibit fungal growth, but little is known about how fungi could interfere in bacterial development in CF lungs. Scedosporium and Lomentospora species present peptidorhamnomannans (PRMs) in their cell wall that play crucial roles in fungal adhesion and interaction with host epithelial cells and the immune system. The present study aimed to analyze whether PRMs extracted from Lomentospora prolificans, Scedosporium apiospermum, Scedosporium boydii, and Scedosporium aurantiacum block bacterial growth and biofilm formation in vitro. PRM from L. prolificans and S. boydii displayed the best bactericidal effect against methicillin resistant Staphylococcus aureus (MRSA), Burkholderia cepacia, and Escherichia coli, but not Pseudomonas aeruginosa, all of which are the most frequently found bacteria in CF lungs. In addition, biofilm formation was inhibited in all bacteria tested using PRMs at minimal inhibitory concentration (MIC). These results suggest that PRMs from the Scedosporium and Lomentospora surface seem to play an important role in Scedosporium colonization in CF patients, helping to clarify how these pathogens interact to each other in CF lungs.

Pseudomonas aeruginosa (Pa) is the leading cause of chronic lung infection in Cystic Fibrosis (CF) patients. It is well recognized that CF epithelial cells fail to develop an appropriate response to infection, allowing bacterial colonization and a chronic inflammatory response. Since long non-coding RNAs (lncRNAs), are known to play a key role in regulating mammalian innate immune response, we hypothesized that CF cells exposed to Pa could express a specific lncRNA signature responsible of the maladaptative CF response. We analyzed transcriptomic datasets to compare the expression profiles of lncRNAs in primary CF and non-CF epithelial cells infected with Pa at 0, 2, 4, and 6 h of infection. Our analysis identified temporal expression signatures of 25, 73, 15, and 26 lncRNA transcripts differentially expressed at 0, 2, 4, and 6 h post-infection respectively, between CF and non-CF cells. In addition, we identified profiles specific to CF and non-CF cells. The differential expression of two candidate lncRNAs were independently validated using real-time PCR. We identified a specific CF signature of lncRNA expression in a context of Pa infection that could potentially play a role in the maladaptive immune response of CF patients.

Cystic fibrosis (CF) disease provokes the accumulation of thick and viscous sputum in the lungs, favoring the development of chronic and polymicrobial infections. Pseudomonas aeruginosa is the main bacterium responsible for these chronic infections, and much of the difficulty involved in eradicating it is due to biofilm formation. However, this could be mitigated using adjuvant compounds that help or potentiate the antibiotic action. Therefore, the main goal of this study was to search for substances that function as adjuvants and also as biofilm-controlling compounds, preventing or dismantling P. aeruginosa biofilms formed in an in vitro CF airway environment. Dual combinations of compounds with subinhibitory (1 and 2 mg/L) and inhibitory concentrations (4 mg/L) of ciprofloxacin were tested to inhibit the bacterial growth and biofilm formation (prophylactic approach) and to eradicate 24-h-old P. aeruginosa populations, including planktonic cells and biofilms (treatment approach). Our results revealed that aspartic acid (Asp) and succinic acid (Suc) restored ciprofloxacin action against P. aeruginosa. Suc combined with 2 mg/L of ciprofloxacin (Suc-Cip) was able to eradicate bacteria, and Asp combined with 4 mg/L of ciprofloxacin (Asp-Cip) seemed to eradicate the whole 24-h-old populations, including planktonic cells and biofilms. Based on biomass depletion data, we noted that Asp induced cell death and Suc seemed somehow to block or reduce the expression of ciprofloxacin resistance. As far as we know, this kind of action had not been reported up till now. The presence of Staphylococcus aureus and Burkholderia cenocepacia did not affect the efficacy of the Asp-Cip and Suc-Cip therapies against P. aeruginosa and, also important, P. aeruginosa depletion from polymicrobial communities did not create a window of opportunity for these species to thrive. Rather the contrary, Asp and Suc also improved ciprofloxacin action against B. cenocepacia. Further studies on the cytotoxicity using lung epithelial cells indicated toxicity of Suc-Cip caused by the Suc. In conclusion, we provided evidences that Asp and Suc could be potential ciprofloxacin adjuvants to eradicate P. aeruginosa living within polymicrobial communities. Asp-Cip and Suc-Cip could be promising therapeutic options to cope with CF treatment failures.

Azole-resistant Aspergillus fumigatus (ARAf) has emerged worldwide during the last decades. Drug pressure after long term treatments of chronically infected patients and the propagation of environmental clones selected under the pressure of imidazoles fungicides used in agriculture and farming both account for this emergence. The objectives of this study were to determine the rate of azole resistance in Aspergillus fumigatus during a 5-year period, taking into account (i) differences between underlying diseases of the patients treated, (ii) cross-resistance between azoles, and (iii) focusing on the 5-year evolution of our center's cystic fibrosis cohort. Overall, the rates of voriconazole (VRC)-resistant and itraconazole (ITC)-resistant A. fumigatus isolates were 4.1% (38/927) and 14.5% (95/656), respectively, corresponding to 21/426 (4.9%) and 44/308 (14.3%) patients, respectively. Regarding cross-resistance, among VRC-R isolates tested for ITC, nearly all were R (20/21;95%), compared to only 27% (20/74) of VRC-R among ITC-R isolates. The level of azole resistance remained somewhat stable over years but greatly varied according to the azole drug, patient origin, and clinical setting. Whereas azole resistance during invasive aspergillosis was very scarce, patients with cystic fibrosis were infected with multiple strains and presented the highest rate of resistance: 5% (27/539) isolates were VRC-R and 17.9% (78/436) were ITC-R. These results underline that the interpretation of the azole resistance level in Aspergilllus fumigatus in a routine setting may consider the huge variability depending on the azole drug, the clinical setting, the patient background and the type of 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.

Background: Rabbits are useful for preclinical studies of sinusitis because of similar physiologic features to humans. The objective of this study is to develop a rabbit model of sinusitis that permits assessment of microanatomy and sampling for evaluating shifts in the sinus microbiota during the development of sinusitis and to test how the mucociliary clearance (MCC) defect might lead to dysbiosis and chronic rhinosinusitis (CRS). Methods: Generation of CRS was accomplished with an insertion of a sterile sponge into the left middle meatus of New Zealand white rabbits (n = 9) for 2 weeks. After sponge removal, 4 rabbits were observed for another 10 weeks and evaluated for CRS using endoscopy, microCT, visualization of the functional micro-anatomy by micro-optical coherence tomography (μOCT), and histopathological analysis of the sinus mucosa. Samples were taken from the left middle meatus and submitted for microbiome analysis. Results: CT demonstrated opacification of all left sinuses at 2 weeks in all rabbits (n = 9), which persisted in animals followed for another 12 weeks (n = 4). Histology at week 2 showed mostly neutrophils. On week 14, significant infiltration of plasma cells and lymphocytes was noted with increased submucosal glands compared to controls (p = 0.02). Functional microanatomy at 2 weeks showed diminished periciliary layer (PCL) depth (p < 0.0001) and mucus transport (p = 0.0044) compared to controls despite a thick mucus layer. By 12 weeks, the thickened mucus layer was resolved but PCL depletion persisted in addition to decreased ciliary beat frequency (CBF; p < 0.0001). The mucin fermenting microbes (Lactobacillales, Bacteroidales) dominated on week 2 and there was a significant shift to potential pathogens (e.g., Pseudomonas, Burkholderia) by week 14 compared to both controls and the acute phase (p < 0.05). Conclusion: We anticipate this reproducible model will provide a means for identifying underlying mechanisms of airway-surface liquid (ASL) depletion and fundamental changes in sinus microbial communities that contribute to the development of CRS. The rabbit model of sinusitis exhibited diminished PCL depth with delayed mucus transport and significant alterations and shift in the sinus microbiome during the development of chronic inflammation.

Background: Chronic rhinosinusitis (CRS) is characterized by complex bacterial infections with persistent inflammation. Based on our rabbit model of sinusitis, blockage of sinus ostia generated a shift in microbiota to a predominance of mucin degrading microbes (MDM) with acute inflammation at 2 weeks. This was followed by conversion to chronic sinus inflammation at 3 months with a robust increase in pathogenic bacteria (e.g., Pseudomonas). MDMs are known to produce acid metabolites [short chain fatty acids (SCFA)] that have the potential to stimulate pathogen growth by offering a carbon source to non-fermenting sinus pathogens (e.g., Pseudomonas). The objective of this study is to evaluate the concentrations of SCFA within the mucus and its contribution to the growth of P. aeruginosa. Methods: Healthy and sinusitis mucus from the rabbit model were collected and co-cultured with the PAO1 strain of P. aeruginosa for 72 h and colony forming units (CFUs) were determined with the targeted quantification of three SCFAs (acetate, propionate, butyrate). Quantification of SCFAs in healthy and sinusitis mucus from patients with P. aeruginosa was also performed via high performance liquid chromatography. Results: To provide evidence of fermentative activity, SCFAs were quantified within the mucus samples from rabbits with and without sinusitis. Acetate concentrations were significantly greater in sinusitis mucus compared to controls (4.13 ± 0.53 vs. 1.94 ± 0.44 mM, p < 0.01). After 72 h of co-culturing mucus samples with PAO1 in the presence of mucin medium, the blue-green pigment characteristic of Pseudomonas was observed throughout tubes containing sinusitis mucus. CFUs were higher in cultures containing mucus samples from sinusitis (8.4 × 109 ± 4.8 × 107) compared to control (1.4 × 109 ± 2.0 × 107) or no mucus (1.5 × 109 ± 2.1 × 107) (p < 0.0001). To provide evidence of fermentative activity in human CRS with P. aeruginosa, the presence of SCFAs in human mucus was analyzed and all SCFAs were significantly higher in CRS with P. aeruginosa compared to controls (p < 0.05). Conclusion: Given that SCFAs are solely derived from bacterial fermentation, our evidence suggests a critical role for mucin-degrading bacteria in generating carbon-source nutrients for pathogens. MDM may contribute to the development of recalcitrant CRS by degrading mucins, thus providing nutrients for potential pathogens like P. aeruginosa.

Cystic fibrosis (CF) lung disease is aggravated by recurrent and ultimately chronic bacterial infections. One of the key pathogens in adult CF lung disease is P. aeruginosa (PA). In addition to bacteria, respiratory viral infections are suggested to trigger pulmonary exacerbations in CF. To date, little is known on how chronic infections with PA influence susceptibility and response to viral infection. We investigated the interactions between PA, human rhinovirus (HRV) and the airway epithelium in a model of chronic PA infection using differentiated primary bronchial epithelial cells (pBECs) and clinical PA isolates obtained from the respiratory sample of a CF patient. Cells were repeatedly infected with either a mucoid or a non-mucoid PA isolate for 16 days to simulate chronic infection, and subsequently co-infected with HRV. Key cytokines and viral RNA were quantified by cytometric bead array, ELISA and qPCR. Proteolytic degradation of IL-6 was analyzed by Western Blots. Barrier function was assessed by permeability tests and transepithelial electric resistance measurements. Virus infection stimulated the production of inflammatory and antiviral mediators, including interleukin (IL)-6, CXCL-8, tumor necrosis factor (TNF)-α, and type I/III interferons. Co-infection with a non-mucoid PA isolate increased IL-1β protein concentrations (28.88 pg/ml vs. 6.10 pg/ml), but in contrast drastically diminished levels of IL-6 protein (53.17 pg/ml vs. 2301.33 pg/ml) compared to virus infection alone. Conditioned medium obtained from co-infections with a non-mucoid PA isolate and HRV was able to rapidly degrade recombinant IL-6 in a serine protease-dependent manner, whereas medium from individual infections or co-infections with a mucoid isolate had no such effect. After co-infection with HRV and the non-mucoid PA isolate, we detected lower mRNA levels of Forkhead box J1 (FOXJ1) and Cilia Apical Structure Protein (SNTN), markers of epithelial cell differentiation to ciliated cells. Moreover, epithelial permeability was increased and barrier function compromised compared to single infections. These data show that PA infection can influence the response of bronchial epithelial cells to viral infection. Altered innate immune responses and compromised epithelial barrier function may contribute to an aggravated course of viral infection in PA-infected airways.