Aberrant immune programming in neutrophils in cystic fibrosis.

Cystic fibrosis is a life-shortening genetic disorder, caused by mutations in the gene that encodes cystic fibrosis transmembrane-conductance regulator, a cAMP-activated chloride and bicarbonate channel. Persistent neutrophilic inflammation is a major contributor to cystic fibrosis lung disease. However, how cystic fibrosis transmembrane-conductance regulator loss of function leads to excessive inflammation and its clinical sequela remains incompletely understood. In this study, neutrophils from F508del-CF and healthy control participants were compared for gene transcription. We found that cystic fibrosis circulating neutrophils have a prematurely primed basal state with significantly higher scores for activation, chemotaxis, immune signaling, and pattern recognition. Such an irregular basal state appeared not related to the blood environment and was also observed in neutrophils derived from the F508del-CF HL-60 cell line, indicating an innate characteristic of the phenotype. Lipopolysaccharides (LPS) stimulation drastically shifted the transcriptional landscape of healthy control neutrophils toward a robust immune response; however, cystic fibrosis neutrophils were immune-exhausted, reflected by abnormal cell aging and fate determination in gene programming. Moreover, cystic fibrosis sputum neutrophils differed significantly from cystic fibrosis circulating neutrophils in gene transcription with increased inflammatory response, aging, apoptosis, and necrosis, suggesting additional environmental influences on the neutrophils in cystic fibrosis lungs. Taken together, our data indicate that loss of cystic fibrosis transmembrane-conductance regulator function has intrinsic effects on neutrophil immune programming, leading to premature priming and dysregulated response to challenge.

Loss of CFTR function in macrophages alters the cell transcriptional program and delays lung resolution of inflammation.

Cystic fibrosis (CF) is an autosomal recessive genetic disorder caused by mutations in the CF Transmembrane-conductance Regulator (CFTR) gene. The most severe pathologies of CF occur in the lung, manifesting as chronic bacterial infection, persistent neutrophilic inflammation, and mucopurulent airway obstruction. Despite increasing knowledge of the CF primary defect and the resulting clinical sequelae, the relationship between the CFTR loss of function and the neutrophilic inflammation remains incompletely understood. Here, we report that loss of CFTR function in macrophages causes extended lung inflammation. After intratracheal inoculation with Pseudomonas aeruginosa, mice with a macrophage-specific Cftr-knockout (Mac-CF) were able to mount an effective host defense to clear the bacterial infection. However, three days post-inoculation, Mac-CF lungs demonstrated significantly more neutrophil infiltration and higher levels of inflammatory cytokines, suggesting that Mac-CF mice had a slower resolution of inflammation. Single-cell RNA sequencing revealed that absence of CFTR in the macrophages altered the cell transcriptional program, affecting the cell inflammatory and immune responses, antioxidant system, and mitochondrial respiration. Thus, loss of CFTR function in macrophages influences cell homeostasis, leading to a dysregulated cellular response to infection that may exacerbate CF lung disease.

Genome Editing for Cystic Fibrosis.

Cystic fibrosis (CF) is a monogenic recessive genetic disorder caused by mutations in the CF Transmembrane-conductance Regulator gene (CFTR). Remarkable progress in basic research has led to the discovery of highly effective CFTR modulators. Now ~90% of CF patients are treatable. However, these modulator therapies are not curative and do not cover the full spectrum of CFTR mutations. Thus, there is a continued need to develop a complete and durable therapy that can treat all CF patients once and for all. As CF is a genetic disease, the ultimate therapy would be in-situ repair of the genetic lesions in the genome. Within the past few years, new technologies, such as CRISPR/Cas gene editing, have emerged as an appealing platform to revise the genome, ushering in a new era of genetic therapy. This review provided an update on this rapidly evolving field and the status of adapting the technology for CF therapy.

Neutrophil defect and lung pathogen selection in cystic fibrosis.

Cystic fibrosis is a life-threatening genetic disorder caused by mutations in the CFTR chloride channel. Clinically, over 90% of patients with cystic fibrosis succumb to pulmonary complications precipitated by chronic bacterial infections, predominantly by Pseudomonas aeruginosa and Staphylococcus aureus. Despite the well-characterized gene defect and clearly defined clinical sequelae of cystic fibrosis, the critical link between the chloride channel defect and the host defense failure against these specific pathogens has not been established. Previous research from us and others has uncovered that neutrophils from patients with cystic fibrosis are defective in phagosomal production of hypochlorous acid, a potent microbicidal oxidant. Here we report our studies to investigate if this defect in hypochlorous acid production provides P. aeruginosa and S. aureus with a selective advantage in cystic fibrosis lungs. A polymicrobial mixture of cystic fibrosis pathogens (P. aeruginosa and S. aureus) and non-cystic fibrosis pathogens (Streptococcus pneumoniae, Klebsiella pneumoniae, and Escherichia coli) was exposed to varied concentrations of hypochlorous acid. The cystic fibrosis pathogens withstood higher concentrations of hypochlorous acid than did the non-cystic fibrosis pathogens. Neutrophils derived from F508del-CFTR HL-60 cells killed P. aeruginosa less efficiently than did the wild-type counterparts in the polymicrobial setting. After intratracheal challenge in wild-type and cystic fibrosis mice, the cystic fibrosis pathogens outcompeted the non-cystic fibrosis pathogens and exhibited greater survival in the cystic fibrosis lungs. Taken together, these data indicate that reduced hypochlorous acid production due to the absence of CFTR function creates an environment in cystic fibrosis neutrophils that provides a survival advantage to specific microbes-namely, S. aureus and P. aeruginosa-in the cystic fibrosis lungs.

ABERRANT IMMUNE PROGRAMMING IN NEUTROPHILS IN CYSTIC FIBROSIS.

Cystic fibrosis (CF) is a life-shortening genetic disorder, caused by mutations in the gene that encodes Cystic Fibrosis Transmembrane-conductance Regulator (CFTR), a cAMP-activated chloride and bicarbonate channel. Although multiple organ systems can be affected, CF lung disease claims the most morbidity and mortality due to chronic bacterial infection, persistent neutrophilic inflammation, and mucopurulent airway obstruction. Despite the clear predominance of neutrophils in these pathologies, how CFTR loss-of-function affects these cells per se remains incompletely understood. Here, we report the profiling and comparing of transcriptional signatures of peripheral blood neutrophils from CF participants and healthy human controls (HC) at the single-cell level. Circulating CF neutrophils had an aberrant basal state with significantly higher scores for activation, chemotaxis, immune signaling, and pattern recognition, suggesting that CF neutrophils in blood are prematurely primed. Such an abnormal basal state was also observed in neutrophils derived from an F508del-CF HL-60 cell line, indicating an innate characteristic of the phenotype. LPS stimulation drastically shifted the transcriptional landscape of HC circulating neutrophils towards a robust immune response, however, CF neutrophils were immune-exhausted. Moreover, CF blood neutrophils differed significantly from CF sputum neutrophils in gene programming with respect to neutrophil activation and aging, as well as inflammatory signaling, highlighting additional environmental influences on the neutrophils in CF lungs. Taken together, loss of CFTR function has intrinsic effects on neutrophil immune programming that leads to premature priming and dysregulated response to challenge.

Cftr deletion in mouse epithelial and immune cells differentially influence the intestinal microbiota.

Cystic fibrosis (CF) is a life-threatening genetic disorder, caused by mutations in the CF transmembrane-conductance regulator gene (cftr) that encodes CFTR, a cAMP-activated chloride and bicarbonate channel. Clinically, CF lung disease dominates the adult patient population. However, its gastrointestinal illness claims the early morbidity and mortality, manifesting as intestinal dysbiosis, inflammation and obstruction. As CF is widely accepted as a disease of epithelial dysfunction, it is unknown whether CFTR loss-of-function in immune cells contributes to these clinical outcomes. Using cftr genetic knockout and bone marrow transplantation mouse models, we performed 16S rRNA gene sequencing of the intestinal microbes. Here we show that cftr deletion in both epithelial and immune cells collectively influence the intestinal microbiota. However, the immune defect is a major factor determining the dysbiosis in the small intestine, while the epithelial defect largely influences that in the large intestine. This finding revises the current concept by suggesting that CF epithelial defect and immune defect play differential roles in CF intestinal disease.

Neutrophil dysfunction in the pathogenesis of cystic fibrosis.

Polymorphonuclear neutrophils (PMNs) figure prominently in host defense against infection and in noninfectious inflammation. Mobilized early in an inflammatory response, PMNs mediate immediate cellular defense against microbes and orchestrate events that culminate in cessation of inflammation and restoration of homeostasis. Failure to terminate the inflammatory response and its causes can fuel exuberant inflammation characteristic of many human diseases, including cystic fibrosis (CF), an autosomal recessive genetic disease caused by mutations in the CF transmembrane conductance regulator. CF affects multiple end organs, with persistent bacterial infection and chronic neutrophilic inflammation in airways predominating the clinical picture. To match the diverse microbial challenges that they may encounter, PMNs possess a variety of antimicrobial systems to slow or kill invading microorganisms confined in their phagosomes. Prominent among PMN defense systems is their ability to generate hypochlorous acid, a potent microbicide, by reacting oxidants generated by the NADPH oxidase with myeloperoxidase (MPO) released from azurophilic granules in the presence of chloride (Cl-). Products of the MPO-H2O2-Cl system oxidize susceptible biomolecules and support robust antimicrobial action against many, but not all, potential human pathogens. Underscoring that the MPO-H2O2-Cl system is integral to optimal host defense and proper regulation of inflammation, individuals with defects in any component of this system, as seen in chronic granulomatous disease or MPO deficiency, incur increased rates or severity of infection and signs of dysregulated inflammatory responses. We focus attention in this review on the molecular basis for and the clinical consequences of defects in the MPO-H2O2-Cl system because of the compromised Cl transport seen in CF. We will discuss first how the MPO-H2O2-Cl system in healthy PMNs participates in host defense and resolution of inflammation and then review how a defective MPO-H2O2-Cl system contributes to the increased susceptibility to infection and dysregulated inflammation associated with the clinical manifestations of CF.

Myeloid CFTR loss-of-function causes persistent neutrophilic inflammation in cystic fibrosis.

Persistent neutrophilic inflammation is a hallmark of cystic fibrosis (CF). However, the mechanisms underlying this outstanding pathology remain incompletely understood. Here, we report that CFTR in myeloid immune cells plays a pivotal role in control of neutrophilic inflammation. Myeloid CFTR-Knockout (Mye-Cftr-/-) mice and congenic wild-type (WT) mice were challenged peritoneally with zymosan particles at different doses, creating aseptic peritonitis with varied severity. A high-dose challenge resulted in significantly higher mortality in Mye-Cftr-/- mice, indicating an intrinsic defect in host control of inflammation in mice whose myeloid cells lack CF. The low-dose challenge demonstrated an impaired resolution of inflammation in Mye-Cftr-/- mice, reflected by a significant overproduction of proinflammatory cytokines, including neutrophil chemokines MIP-2 and KC, and sustained accumulation of neutrophils. Tracing neutrophil mobilization in vivo demonstrated that myeloid CF mice recruited significantly more neutrophils than did WT mice. Pulmonary challenge with zymosan elicited exuberant inflammation in the lung and recapitulated the findings from peritoneal challenge. To determine the major type of cell that was primarily responsible for the over-recruitment of neutrophils, we purified and cultured ex vivo zymosan-elicited peritoneal neutrophils and macrophages. The CF neutrophils produced significantly more MIP-2 than did the WT counterparts, and peripheral blood neutrophils isolated from myeloid CF mice also produced significantly more MIP-2 after zymosan stimulation in vitro. These data altogether suggest that CFTR dysfunction in myeloid immune cells, especially neutrophils, leads to hyperinflammation and excessive neutrophil mobilization in the absence of infection. Thus, dysregulated inflammation secondary to abnormal or absent CFTR in myeloid cells may underlie the clinically observed neutrophilic inflammation in CF.

Efficient Gene Editing at Major CFTR Mutation Loci.

Cystic fibrosis (CF) is a lethal autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Nuclease-mediated precise gene editing (PGE) represents a promising therapy for CF, for which an efficient strategy that is free of viral vector, drug selection, and reporter enrichment (VDR free) is desirable. Here we compared different transfection methods (lipofectamine versus electroporation) and formats (plasmid DNA versus ribonucleoprotein) in delivering the CRISPR/Cas9 elements along with single-stranded oligodeoxynucleotides (ssODNs) to clinically relevant cells targeting major CFTR mutation loci. We demonstrate that, among different combinations, electroporation of CRISPR/Cas9 and guide RNA (gRNA) ribonucleoprotein (Cas9 RNP) is the most effective one. By using this VDR-free method, 4.8% to 27.2% efficiencies were achieved in creating dF508, G542X, and G551D mutations in a wild-type induced pluripotent stem cell (iPSC) line. When it is applied to a patient-derived iPSC line carrying the dF508 mutation, a greater than 20% precise correction rate was achieved. As expected, genetic correction leads to the restoration of CFTR function in iPSC-derived proximal lung organoids, as well as in a patient-derived adenocarcinoma cell line CFPAC-1. The present work demonstrates the feasibility of gene editing-based therapeutics toward monogenic diseases such as CF.

Establishment of a ΔF508-CF promyelocytic cell line for cystic fibrosis research and drug screening.

Cystic fibrosis (CF), one of the most common genetic disorders, is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. In spite of significant improvement in patient life expectancy, the disease remains lethal and incurable. Clinically, CF lung disease claims the most morbidity and mortality, characterized by chronic bacterial infection, persistent neutrophilic inflammation, and purulent small airway obstruction. Although all these manifestations are highly associated with neutrophils, the actual role of this phagocyte in the disease pathogenesis has not been fully appreciated. One of the major obstacles impeding such progress is the lack of CF neutrophil cell lines. Taking advantage of the new CRISPR/Cas9 gene-editing technology, we have generated a homozygous ΔF508-CF promyelocytic cell line from HL-60 cells, from which unlimited CF neutrophil cells can be differentiated. The derived cells showed defective CFTR presentation, deficient phagosomal hypochlorous acid (HOCl) production, and compromised microbial killing. Such a phenotype recapitulates that of primary neutrophils from CF patients. Thus, the established human CF promyelocytic cell line should be a useful tool for future CF basic research and drug screening.

Metabolism of selective 20-epi-vitamin D3 analogs in rat osteosarcoma UMR-106 cells: Isolation and identification of four novel C-1 fatty acid esters of 1α,25-dihydroxy-16-ene-20-epi-vitamin D3.

Analogs of 1α,25-dihydroxyvitamin D3 (S1) with 20-epi modification (20-epi analogs) possess unique biological properties. We previously reported that 1α,25-dihydroxy-20-epi-vitamin D3 (S2), the basic 20-epi analog is metabolized into less polar metabolites (LPMs) in rat osteosarcoma cells (UMR-106) but not in a perfused rat kidney. Furthermore, we also noted that only selective 20-epi analogs are metabolized into LPMs. For example, 1α,25-dihydroxy-16-ene-20-epi-vitamin D3 (S4), but not 1α,25-dihydroxy-16-ene-23-yne-20-epi-vitamin D3 (S5) is metabolized into LPMs. In spite of these novel findings, the unequivocal identification of LPMs has not been achieved to date. We report here on a thorough investigation of the metabolism of S4 in UMR-106 cells and isolated two major LPMs produced directly from the substrate S4 itself and two minor LPMs produced from 3-epi-S4, a metabolite of S4 produced through C-3 epimerization pathway. Using GC/MS, ESI-MS and 1H NMR analysis, we identified all the four LPMs of S4 as 25-hydroxy-16-ene-20-epi-vitamin D3-1-stearate and 25-hydroxy-16-ene-20-epi-vitamin D3-1-oleate and their respective C-3 epimers. We report here for the first time the elucidation of a novel pathway of metabolism in UMR-106 cells in which both 1α,25(OH)2-16-ene-20-epi-D3 and 1α,25(OH)2-16-ene-20-epi-3-epi-D3 undergo C-1 esterification into stearic and oleic acid esters.

CFTR targeting during activation of human neutrophils.

Cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel, plays critical roles in phagocytic host defense. However, how activated neutrophils regulate CFTR channel distribution subcellularly is not well defined. To investigate, we tested multiple Abs against different CFTR domains, to examine CFTR expression in human peripheral blood neutrophils by flow cytometry. The data confirmed that resting neutrophils had pronounced CFTR expression. Activation of neutrophils with soluble or particulate agonists did not significantly increase CFTR expression level, but induced CFTR redistribution to cell surface. Such CFTR mobilization correlated with cell-surface recruitment of formyl-peptide receptor during secretory vesicle exocytosis. Intriguingly, neutrophils from patients with ΔF508-CF, despite expression of the mutant CFTR, showed little cell-surface mobilization upon stimulation. Although normal neutrophils effectively targeted CFTR to their phagosomes, ΔF508-CF neutrophils had impairment in that process, resulting in deficient hypochlorous acid production. Taken together, activated neutrophils regulate CFTR distribution by targeting this chloride channel to the subcellular sites of activation, and ΔF508-CF neutrophils fail to achieve such targeting, thus undermining their host defense function.

Phenotypic and Molecular Characterization of Domestic Cat (Felis catus) Spermatogonial Stem Cells.

In many mammalian species, surface markers have been used to obtain enriched populations of spermatogonial stem cells (SSCs) for assisted reproduction and other applications; however, little is known about the expression patterns of feline SSCs. In this study, we assessed expression of the SSC surface markers commonly used in other species, KIT, ITGA6, CD9, GFRalpha1, ADGRA3, and THY1, in addition to the less frequently used pluripotent markers TRA-1-60, TRA-1-81, SSEA-1, and SSEA-4 in SSCs of both prepubertal and adult domestic cats (Felis catus). To further characterize cat SSCs, we sorted cells using SSC-specific markers and evaluated the expression of the pluripotent transcription factors NANOG, POU5F1, and SOX2 and the proto-oncogene MYC within these populations. We concluded that SSC surface markers used in other mammalian species were not specific for identifying cat SSCs. However, the pluripotent markers we evaluated were more specific to cat spermatogonia, and the presence of SSEA-1 and SSEA-4 in fewer and primarily individual cells suggests that these two markers may be used for enrichment of cat SSCs. The expression of pluripotent transcription factors at mRNA level by single-stained cells positive for SSEA-4 and by dual-stained cells positive for both GFRalpha1 and SSEA-4 reflects the undifferentiated stage of cat SSCs. The absence of transcription factors in double-stained cells positive for only one marker implies the loss of the stem cell-like identity with the loss of either GFRalpha1 or SSEA-4. Further investigation is warranted to elucidate the biological characteristics of these spermatogonial subpopulations.

Characterization and Multilineage Differentiation of Domestic and Black-Footed Cat Mesenchymal Stromal/Stem Cells from Abdominal and Subcutaneous Adipose Tissue.

Transplantation of mesenchymal stem cells (MSCs) isolated from bone marrow or adipose tissue is emerging as a promising tool for cell replacement therapy and regenerative medicine in domestic and endangered animal species. Defining the differentiation capability of adipose-derived mesenchymal stromal/stem cells (AMSCs) collected from different depot sites of adipose tissue will be essential for developing strategies for cell replacement therapy. In the present study, we compared the biological characteristics of domestic cat AMSCs isolated from visceral fat of the abdominal cavity (AB) with AMSCs from subcutaneous (SQ) tissue, and the functional capability of domestic and black-footed cat (Felis nigripes) AMSCs to differentiate into other cell types. Our results showed that both domestic and black-footed cat adipose-derived stromal vascular fractions contained AMSCs. Both domestic cat AB- and SQ-AMSCs showed important clonogenic ability and the minimal MSC immunophenotype as defined by the International Society for Cellular Therapy in humans. However, domestic cat AB-AMSCs had higher percentages of cells positive for MSCs-associated cluster of differentiation (CD) markers CD90(+) and CD105(+) (92% and 80%, respectively) than those of SQ-AMSCs (77% and 58%, respectively). Although these results may suggest that AB-AMSCs may be more multipotent than SQ-AMSCs, both types of cells showed similar expression of pluripotent genes Oct-4 and Klf4, except for higher expression of Nanog than in AB-AMSCs, and equivalent in vitro multilineage differentiation. Under appropriate stimuli, the black-footed cat and both domestic cat AB- and SQ-AMSCs differentiated not only toward mesoderm cell lineages but also toward ectoderm cell lineage, such as neuron cell-like cells. Black-footed cat AMSCs had more capability to differentiate toward chondrocytes. These results suggest that the defined AMSC population (regardless of site of collection) could potentially be employed as a therapeutic agent for both domestic and endangered diseased or injured felids.

Salt, chloride, bleach, and innate host defense.

Salt provides 2 life-essential elements: sodium and chlorine. Chloride, the ionic form of chlorine, derived exclusively from dietary absorption and constituting the most abundant anion in the human body, plays critical roles in many vital physiologic functions, from fluid retention and secretion to osmotic maintenance and pH balance. However, an often overlooked role of chloride is its function in innate host defense against infection. Chloride serves as a substrate for the generation of the potent microbicide chlorine bleach by stimulated neutrophils and also contributes to regulation of ionic homeostasis for optimal antimicrobial activity within phagosomes. An inadequate supply of chloride to phagocytes and their phagosomes, such as in CF disease and other chloride channel disorders, severely compromises host defense against infection. We provide an overview of the roles that chloride plays in normal innate immunity, highlighting specific links between defective chloride channel function and failures in host defense.

NRAV, a long noncoding RNA, modulates antiviral responses through suppression of interferon-stimulated gene transcription.

Long noncoding RNAs (lncRNAs) modulate various biological processes, but their role in host antiviral responses is largely unknown. Here we identify a lncRNA as a key regulator of antiviral innate immunity. Following from the observation that a lncRNA that we call negative regulator of antiviral response (NRAV) was dramatically downregulated during infection with several viruses, we ectopically expressed NRAV in human cells or transgenic mice and found that it significantly promotes influenza A virus (IAV) replication and virulence. Conversely, silencing NRAV suppressed IAV replication and virus production, suggesting that reduction of NRAV is part of the host antiviral innate immune response to virus infection. NRAV negatively regulates the initial transcription of multiple critical interferon-stimulated genes (ISGs), including IFITM3 and MxA, by affecting histone modification of these genes. Our results provide evidence for a lncRNA in modulating the antiviral interferon response.

Neutrophil-mediated phagocytic host defense defect in myeloid Cftr-inactivated mice.

Cystic fibrosis (CF) is a common and deadly inherited disease, caused by mutations in the CFTR gene that encodes a cAMP-activated chloride channel. One outstanding manifestation of the disease is the persistent bacterial infection and inflammation in the lung, which claims over 90% of CF mortality. It has been debated whether neutrophil-mediated phagocytic innate immunity has any intrinsic defect that contributes to the host lung defense failure. Here we compared phagosomal CFTR targeting, hypochlorous acid (HOCl) production, and microbial killing of the neutrophils from myeloid Cftr-inactivated (Myeloid-Cftr-/-) mice and the non-inactivated control (Cftrfl10) mice. We found that the mutant CFTR that lacked Exon-10 failed to target to the neutrophil phagosomes. This dysfunction resulted in impaired intraphagosomal HOCl production and neutrophil microbial killing. In vivo lung infection with a lethal dose of Pseudomonas aeruginosa caused significantly higher mortality in the myeloid CF mice than in the controls. The myeloid-Cftr-/- lungs were deficient in bacterial clearance, and had sustained neutrophilic inflammation and stalled transition from early to late immunity. These manifestations recapitulated the symptoms of human CF lungs. The data altogether suggest that myeloid CFTR expression is critical to normal host lung defense. CFTR dysfunction in neutrophils compromises the phagocytic innate immunity, which may predispose CF lungs to infection.

Cystic fibrosis transmembrane conductance regulator recruitment to phagosomes in neutrophils.

Optimal microbicidal activity of human polymorphonuclear leukocytes (PMN) relies on the generation of toxic agents such as hypochlorous acid (HOCl) in phagosomes. HOCl formation requires H2O2 produced by the NADPH oxidase, myeloperoxidase derived from azurophilic granules, and chloride ion. Chloride transport from cytoplasm into phagosomes requires chloride channels which include cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel. However, the phagosomal targeting of CFTR in PMN has not been defined. Using human peripheral blood PMN, we determined that 95-99% of lysosomal-associated membrane protein 1 (LAMP-1)-positive mature phagosomes were CFTR positive, as judged by immunostaining and flow cytometric analysis. To establish a model cell system to evaluate CFTR phagosomal recruitment, we stably expressed enhanced green fluorescent protein (EGFP) alone, EGFP-wt-CFTR and EGFP-DF508-CFTR fusion proteins in promyelocytic PLB-985 cells, respectively. After differentiation into neutrophil-like cells, CFTR presentation to phagosomes was examined. EGFP-wt-CFTR was observed to associate with phagosomes and colocalize with LAMP-1. Flow cytometric analysis of the isolated phagosomes indicated that such a phagosomal targeting was determined by the CFTR portion of the fusion protein. In contrast, significantly less EGFP-DF508-CFTR was found in phagosomes, indicating a defective targeting of the molecule to the organelle. Importantly, the CFTR corrector compound VRT-325 facilitated the recruitment of DF508-CFTR to phagosomes. These data demonstrate the possibility of pharmacologic correction of impaired recruitment of mutant CFTR, thereby providing a potential means to augment chloride supply to the phagosomes of PMN in patients with cystic fibrosis to enhance their microbicidal function.

Response of Differentiated Human Airway Epithelia to Alcohol Exposure and Klebsiella Pneumoniae Challenge.

Alcohol abuse has been associated with increased susceptibility to pulmonary infection. It is not fully defined how alcohol contributes to the host defense compromise. Here primary human airway epithelial cells were cultured at an air-liquid interface to form a differentiated and polarized epithelium. This unique culture model allowed us to closely mimic lung infection in the context of alcohol abuse by basolateral alcohol exposure and apical live bacterial challenge. Application of clinically relevant concentrations of alcohol for 24 hours did not significantly alter epithelial integrity or barrier function. When apically challenged with viable Klebsiella pneumoniae, the cultured epithelia had an enhanced tightness which was unaffected by alcohol. Further, alcohol enhanced apical bacterial growth, but not bacterial binding to the cells. The cultured epithelium in the absence of any treatment or stimulation had a base-level IL-6 and IL-8 secretion. Apical bacterial challenge significantly elevated the basolateral secretion of inflammatory cytokines including IL-2, IL-4, IL-6, IL-8, IFN-γ, GM-CSF, and TNF-α. However, alcohol suppressed the observed cytokine burst in response to infection. Addition of adenosine receptor agonists negated the suppression of IL-6 and TNF-α. Thus, acute alcohol alters the epithelial cytokine response to infection, which can be partially mitigated by adenosine receptor agonists.

Chloride transport in functionally active phagosomes isolated from Human neutrophils.

Chloride anion is critical for hypochlorous acid (HOCl) production and microbial killing in neutrophil phagosomes. However, the molecular mechanism by which this anion is transported to the organelle is poorly understood. In this report, membrane-enclosed and functionally active phagosomes were isolated from human neutrophils by using opsonized paramagnetic latex microspheres and a rapid magnetic separation method. The phagosomes recovered were highly enriched for specific protein markers associated with this organelle such as lysosomal-associated membrane protein-1, myeloperoxidase (MPO), lactoferrin, and NADPH oxidase. When FITC-dextran was included in the phagocytosis medium, the majority of the isolated phagosomes retained the fluorescent label after isolation, indicative of intact membrane structure. Flow cytometric measurement of acridine orange, a fluorescent pH indicator, in the purified phagosomes demonstrated that the organelle in its isolated state was capable of transporting protons to the phagosomal lumen via the vacuolar-type ATPase proton pump (V-ATPase). When NADPH was supplied, the isolated phagosomes constitutively oxidized dihydrorhodamine 123, indicating their ability to produce hydrogen peroxide. The preparations also showed a robust production of HOCl within the phagosomal lumen when assayed with the HOCl-specific fluorescent probe R19-S by flow cytometry. MPO-mediated iodination of the proteins covalently conjugated to the phagocytosed beads was quantitatively measured. Phagosomal uptake of iodide and protein iodination were significantly blocked by chloride channel inhibitors, including CFTRinh-172 and NPPB. Further experiments determined that the V-ATPase-driving proton flux into the isolated phagosomes required chloride cotransport, and the cAMP-activated CFTR chloride channel was a major contributor to the chloride transport. Taken together, the data suggest that the phagosomal preparation described herein retains ion transport properties, and multiple chloride channels including CFTR are responsible for chloride supply to neutrophil phagosomes.