Single-Cell Transcriptional Archetypes of Airway Inflammation in Cystic Fibrosis.

Rationale: Cystic fibrosis (CF) is a life-shortening, multisystem hereditary disease caused by abnormal chloride transport. CF lung disease is driven by innate immune dysfunction and exaggerated inflammatory responses that contribute to tissue injury. To define the transcriptional profile of this airway immune dysfunction, we performed the first single-cell transcriptome characterization of CF sputum.Objectives: To define the transcriptional profile of sputum cells and its implication in the pathogenesis of immune function and the development of CF lung disease.Methods: We performed single-cell RNA sequencing of sputum cells from nine subjects with CF and five healthy control subjects. We applied novel computational approaches to define expression-based cell function and maturity profiles, herein called transcriptional archetypes.Measurements and Main Results: The airway immune cell repertoire shifted from alveolar macrophages in healthy control subjects to a predominance of recruited monocytes and neutrophils in CF. Recruited lung mononuclear phagocytes were abundant in CF and were separated into the following three archetypes: activated monocytes, monocyte-derived macrophages, and heat shock-activated monocytes. Neutrophils were the most prevalent in CF, with a dominant immature proinflammatory archetype. Although CF monocytes exhibited proinflammatory features, both monocytes and neutrophils showed transcriptional evidence of abnormal phagocytic and cell-survival programs.Conclusions: Our findings offer an opportunity to understand subject-specific immune dysfunction and its contribution to divergent clinical courses in CF. As we progress toward personalized applications of therapeutic and genomic developments, we hope this inflammation-profiling approach will enable further discoveries that change the natural history of CF lung disease.

Cystic fibrosis transmembrane conductance receptor modulator therapy in cystic fibrosis, an update.

PURPOSE OF REVIEW: Cystic fibrosis transmembrane conductance receptor (CFTR) modulators are a new class of drugs that treat the underlying cause of cystic fibrosis. To date, there are four approved medications, which are mutation-specific. Although the number of mutations that respond to these agents is expanding, effective CFTR modulators are not available to all cystic fibrosis patients. The purpose of this article is to review the approved CFTR modulators and discuss the mutations that can be treated with these agents, as well as, review the long-term benefits of modulator therapy. RECENT FINDINGS: More people with cystic fibrosis can be effectively treated with CFTR modulators. The new, highly effective triple therapy, elexacaftor/tezacaftor/ivacaftor is indicated for more than 90% of patients with cystic fibrosis and ivacaftor is now approved for children as young as 6 months of age with 1 of 30 CFTR mutations. Long-term use of modulator therapy is associated with fewer pulmonary exacerbations, maintenance of lung function, improved weight gain, and quality of life. SUMMARY: CFTR modulators are the first therapies developed to treat the underlying defect in cystic fibrosis. Their use is associated with preserved lung function and improved health in patients with cystic fibrosis.

Loss of Cystic Fibrosis Transmembrane Regulator Impairs Intestinal Oxalate Secretion.

Patients with cystic fibrosis have an increased incidence of hyperoxaluria and calcium oxalate nephrolithiasis. Net intestinal absorption of dietary oxalate results from passive paracellular oxalate absorption as modified by oxalate back secretion mediated by the SLC26A6 oxalate transporter. We used mice deficient in the cystic fibrosis transmembrane conductance regulator gene (Cftr) to test the hypothesis that SLC26A6-mediated oxalate secretion is defective in cystic fibrosis. We mounted isolated intestinal tissue from C57BL/6 (wild-type) and Cftr-/- mice in Ussing chambers and measured transcellular secretion of [14C]oxalate. Intestinal tissue isolated from Cftr-/- mice exhibited significantly less transcellular oxalate secretion than intestinal tissue of wild-type mice. However, glucose absorption, another representative intestinal transport process, did not differ in Cftr-/- tissue. Compared with wild-type mice, Cftr-/- mice showed reduced expression of SLC26A6 in duodenum by immunofluorescence and Western blot analysis. Furthermore, coexpression of CFTR stimulated SLC26A6-mediated Cl--oxalate exchange in Xenopus oocytes. In association with the profound defect in intestinal oxalate secretion, Cftr-/- mice had serum and urine oxalate levels 2.5-fold greater than those of wild-type mice. We conclude that defective intestinal oxalate secretion mediated by SLC26A6 may contribute to the hyperoxaluria observed in this mouse model of cystic fibrosis. Future studies are needed to address whether similar mechanisms contribute to the increased risk for calcium oxalate stone formation observed in patients with cystic fibrosis.

Increased susceptibility of Cftr-/- mice to LPS-induced lung remodeling.

Cystic fibrosis (CF) is caused by homozygous mutations of the CF transmembrane conductance regulator (CFTR) Cl(-) channel, which result in chronic pulmonary infection and inflammation, the major cause of morbidity and mortality. Although these processes are clearly related to each other, each is likely to contribute to the pathology differently. Understanding the contribution of each of these processes to the overall pathology has been difficult, because they are usually so intimately connected. Various CF mouse models have demonstrated abnormal immune responses compared with wild-type (WT) littermates when challenged with live bacteria or bacterial products acutely. However, these studies have not investigated the consequences of persistent inflammation on lung tissue in CF mice, which may better model the lung pathology in patients. We characterized the lung pathology and immune response of Cftr(-/-) (CF) and Cftr(+/+) (WT) mice to chronic administration of Pseudomonas aeruginosa lipopolysaccharide (LPS). We show that, after long-term repeated LPS exposure, CF mice develop an abnormal and persistent immune response, which is associated with more robust structural changes in the lung than those observed in WT mice. Although CF mice and their WT littermates develop lung pathology after chronic exposure to LPS, the inflammation and damage resolve in WT mice. However, CF mice do not recover efficiently, and, as a consequence of their chronic inflammation, CF mice are more susceptible to morphological changes and lung remodeling. This study shows that chronic inflammation alone contributes significantly to aspects of CF lung pathology.

Reduced caveolin-1 promotes hyperinflammation due to abnormal heme oxygenase-1 localization in lipopolysaccharide-challenged macrophages with dysfunctional cystic fibrosis transmembrane conductance regulator.

We have previously reported that TLR4 signaling is increased in LPS-stimulated cystic fibrosis (CF) macrophages (MΦs), contributing to the robust production of proinflammatory cytokines. The heme oxygenase-1 (HO-1)/CO pathway modulates cellular redox status, inflammatory responses, and cell survival. The HO-1 enzyme, together with the scaffold protein caveolin 1 (CAV-1), also acts as a negative regulator of TLR4 signaling in MΦs. In this study, we demonstrate that in LPS-challenged CF MΦs, HO-1 does not compartmentalize normally to the cell surface and instead accumulates intracellularly. The abnormal HO-1 localization in CF MΦs in response to LPS is due to decreased CAV-1 expression, which is controlled by the cellular oxidative state, and is required for HO-1 delivery to the cell surface. Overexpression of HO-1 or stimulating the pathway with CO-releasing molecules enhances CAV-1 expression in CF MΦs, suggesting a positive-feed forward loop between HO-1/CO induction and CAV-1 expression. These manipulations re-established HO-1 and CAV-1 cell surface localization in CF MΦs. Consistent with restoration of HO-1/CAV-1-negative regulation of TLR4 signaling, genetic or pharmacological (CO-releasing molecule 2) induced enhancement of this pathway decreased the inflammatory response of CF MΦs and CF mice treated with LPS. In conclusion, our results demonstrate that the counterregulatory HO-1/CO pathway, which is critical in balancing and limiting the inflammatory response, is defective in CF MΦs through a CAV-1-dependent mechanism, exacerbating the CF MΦ response to LPS. This pathway could be a potential target for therapeutic intervention for CF lung disease.

Myosin Ia is required for CFTR brush border membrane trafficking and ion transport in the mouse small intestine.

In enterocytes of the small intestine, endocytic trafficking of CFTR channels from the brush border membrane (BBM) to the subapical endosomes requires the minus-end motor, myosin VI (Myo6). The subapical localization of Myo6 is dependent on myosin Ia (Myo1a) the major plus-end motor associated with the BBM, suggestive of functional synergy between these two motors. In villus enterocytes of the Myo1a KO mouse small intestine, CFTR accumulated in syntaxin-3 positive subapical endosomes, redistributed to the basolateral domain and was absent from the BBM. In colon, where villi are absent and Myo1a expression is low, CFTR exhibited normal localization to the BBM in the Myo1a KO similar to WT. cAMP-stimulated CFTR anion transport in the small intestine was reduced by 58% in the KO, while anion transport in the colon was comparable to WT. Co-immunoprecipitation confirmed the association of CFTR with Myo1a. These data indicate that Myo1a is an important regulator of CFTR traffic and anion transport in the BBM of villus enterocytes and suggest that Myo1a may power apical CFTR movement into the BBM from subapical endosomes. Alternatively, it may anchor CFTR channels in the BBM of villus enterocytes as was proposed for Myo1a's role in BBM localization of sucrase-isomaltase.

Enhancing in vivo cell and tissue targeting by modulation of polymer nanoparticles and macrophage decoys.

The in vivo efficacy of polymeric nanoparticles (NPs) is dependent on their pharmacokinetics, including time in circulation and tissue tropism. Here we explore the structure-function relationships guiding physiological fate of a library of poly(amine-co-ester) (PACE) NPs with different compositions and surface properties. We find that circulation half-life as well as tissue and cell-type tropism is dependent on polymer chemistry, vehicle characteristics, dosing, and strategic co-administration of distribution modifiers, suggesting that physiological fate can be optimized by adjusting these parameters. Our high-throughput quantitative microscopy-based platform to measure the concentration of nanomedicines in the blood combined with detailed biodistribution assessments and pharmacokinetic modeling provides valuable insight into the dynamic in vivo behavior of these polymer NPs. Our results suggest that PACE NPs-and perhaps other NPs-can be designed with tunable properties to achieve desired tissue tropism for the in vivo delivery of nucleic acid therapeutics. These findings can guide the rational design of more effective nucleic acid delivery vehicles for in vivo applications.

Abnormal trafficking and degradation of TLR4 underlie the elevated inflammatory response in cystic fibrosis.

Morbidity and mortality in cystic fibrosis (CF) are due not only to abnormal epithelial cell function, but also to an abnormal immune response. We have shown previously that macrophages lacking CF transmembrane conductance regulator (CFTR), the gene mutated in CF, contribute significantly to the hyperinflammatory response observed in CF. In this study, we show that lack of functional CFTR in murine macrophages causes abnormal TLR4 subcellular localization. Upon LPS stimulation, CFTR macrophages have prolonged TLR4 retention in the early endosome and reduced translocation into the lysosomal compartment. This abnormal TLR4 trafficking leads to increased LPS-induced activation of the NF-κB, MAPK, and IFN regulatory factor-3 pathways and decreased TLR4 degradation, which affects downregulation of the proinflammatory state. In addition to primary murine cells, mononuclear cells isolated from CF patients demonstrate similar defects in response to LPS. Moreover, specific inhibition of CFTR function induces abnormal TLR4 trafficking and enhances the inflammatory response of wild-type murine cells to LPS. Thus, functional CFTR in macrophages influences TLR4 spatial and temporal localization and perturbs LPS-mediated signaling in both murine CF models and patients with CF.

Mouse cystic fibrosis transmembrane conductance regulator forms cAMP-PKA-regulated apical chloride channels in cortical collecting duct.

The cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in many segments of the mammalian nephron, where it may interact with and modulate the activity of a variety of apical membrane proteins, including the renal outer medullary potassium (ROMK) K(+) channel. However, the expression of CFTR in apical cell membranes or its function as a Cl(-) channel in native renal epithelia has not been demonstrated. Here, we establish that CFTR forms protein kinase A (PKA)-activated Cl(-) channels in the apical membrane of principal cells from the cortical collecting duct obtained from mice. These Cl(-) channels were observed in cell-attached apical patches of principal cells after stimulation by forskolin/3-isobutyl-1-methylxanthine. Quiescent Cl(-) channels were present in patches excised from untreated tubules because they could be activated after exposure to Mg-ATP and the catalytic subunit of PKA. The single-channel conductance, kinetics, and anion selectivity of these Cl(-) channels were the same as those of recombinant mouse CFTR channels expressed in Xenopus laevis oocytes. The CFTR-specific closed-channel blocker CFTR(inh)-172 abolished apical Cl(-) channel activity in excised patches. Moreover, apical Cl(-) channel activity was completely absent in principal cells from transgenic mice expressing the DeltaF508 CFTR mutation but was present and unaltered in ROMK-null mice. We discuss the physiologic implications of open CFTR Cl(-) channels on salt handling by the collecting duct and on the functional CFTR-ROMK interactions in modulating the metabolic ATP-sensing of ROMK.

Non-Modulator Therapies: Developing a Therapy for Every Cystic Fibrosis Patient.

Cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapy brings hope to most patients with cystic fibrosis (CF), but not all. For approximately 12% of CF patients with premature termination codon mutations, large deletions, insertions, and frameshifts, the CFTR modulator therapy is not effective. Many believe that genetic-based therapies such as RNA therapies, DNA therapies, and gene editing technologies will be needed to treat mutations that are not responsive to modulator therapy. Delivery of these therapeutic agents to affected cells is the major challenge that will need to be overcome if we are to harness the power of these emerging therapies for the treatment of CF.

Recruitment of monocytes primed to express heme oxygenase-1 ameliorates pathological lung inflammation in cystic fibrosis.

Overwhelming neutrophilic inflammation is a leading cause of lung damage in many pulmonary diseases, including cystic fibrosis (CF). The heme oxygenase-1 (HO-1)/carbon monoxide (CO) pathway mediates the resolution of inflammation and is defective in CF-affected macrophages (MΦs). Here, we provide evidence that systemic administration of PP-007, a CO releasing/O2 transfer agent, induces the expression of HO-1 in a myeloid differentiation factor 88 (MyD88) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)-dependent manner. It also rescues the reduced HO-1 levels in CF-affected cells induced in response to lipopolysaccharides (LPS) or Pseudomonas aeruginosa (PA). Treatment of CF and muco-obstructive lung disease mouse models with a single clinically relevant dose of PP-007 leads to effective resolution of lung neutrophilia and to decreased levels of proinflammatory cytokines in response to LPS. Using HO-1 conditional knockout mice, we show that the beneficial effect of PP-007 is due to the priming of circulating monocytes trafficking to the lungs in response to infection to express high levels of HO-1. Finally, we show that PP-007 does not compromise the clearance of PA in the setting of chronic airway infection. Overall, we reveal the mechanism of action of PP-007 responsible for the immunomodulatory function observed in clinical trials for a wide range of diseases and demonstrate the potential use of PP-007 in controlling neutrophilic pulmonary inflammation by promoting the expression of HO-1 in monocytes/macrophages.

Surface conjugation of antibodies improves nanoparticle uptake in bronchial epithelial cells.

BACKGROUND: Advances in Molecular Therapy have made gene editing through systemic or topical administration of reagents a feasible strategy to treat genetic diseases in a rational manner. Encapsulation of therapeutic agents in nanoparticles can improve intracellular delivery of therapeutic agents, provided that the nanoparticles are efficiently taken up within the target cells. In prior work we had established proof-of-principle that nanoparticles carrying gene editing reagents can mediate site-specific gene editing in fetal and adult animals in vivo that results in functional disease improvement in rodent models of ß-thalassemia and cystic fibrosis. Modification of the surface of nanoparticles to include targeting molecules (e.g. antibodies) holds the promise of improving cellular uptake and specific cellular binding. METHODS AND FINDINGS: To improve particle uptake for diseases of the airway, like cystic fibrosis, our group tested the impact of nanoparticle surface modification with cell surface marker antibodies on uptake in human bronchial epithelial cells in vitro. Binding kinetics of antibodies (Podoplanin, Muc 1, Surfactant Protein C, and Intracellular Adhesion Molecule-1 (ICAM)) were determined to select appropriate antibodies for cellular targeting. The best target-specific antibody among those screened was ICAM antibody. Surface conjugation of nanoparticles with antibodies against ICAM improved cellular uptake in bronchial epithelial cells up to 24-fold. CONCLUSIONS: This is a first demonstration of improved nanoparticle uptake in epithelial cells using conjugation of target specific antibodies. Improved binding, uptake or specificity of particles delivered systemically or to the luminal surface of the airway would potentially improve efficacy, reduce the necessary dose and thus safety of administered therapeutic agents. Incremental improvement in the efficacy and safety of particle-based therapeutic strategies may allow genetic diseases such as cystic fibrosis to be cured on a fundamental genetic level before birth or shortly after birth.

Recruited monocytes/macrophages drive pulmonary neutrophilic inflammation and irreversible lung tissue remodeling in cystic fibrosis.

Persistent neutrophil-dominated lung inflammation contributes to lung damage in cystic fibrosis (CF). However, the mechanisms that drive persistent lung neutrophilia and tissue deterioration in CF are not well characterized. Starting from the observation that, in patients with CF, c-c motif chemokine receptor 2 (CCR2)+ monocytes/macrophages are abundant in the lungs, we investigate the interplay between monocytes/macrophages and neutrophils in perpetuating lung tissue damage in CF. Here we show that CCR2+ monocytes in murine CF lungs drive pathogenic transforming growth factor ß (TGF-ß) signaling and sustain a pro-inflammatory environment by facilitating neutrophil recruitment. Targeting CCR2 to lower the numbers of monocytes in CF lungs ameliorates neutrophil inflammation and pathogenic TGF-ß signaling and prevents lung tissue damage. This study identifies CCR2+ monocytes as a neglected contributor to the pathogenesis of CF lung disease and as a therapeutic target for patients with CF, for whom lung hyperinflammation and tissue damage remain an issue despite recent advances in CF transmembrane conductance regulator (CFTR)-specific therapeutic agents.

In vivo correction of cystic fibrosis mediated by PNA nanoparticles.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. We sought to correct the multiple organ dysfunction of the F508del CF-causing mutation using systemic delivery of peptide nucleic acid gene editing technology mediated by biocompatible polymeric nanoparticles. We confirmed phenotypic and genotypic modification in vitro in primary nasal epithelial cells from F508del mice grown at air-liquid interface and in vivo in F508del mice following intravenous delivery. In vivo treatment resulted in a partial gain of CFTR function in epithelia as measured by in situ potential differences and Ussing chamber assays and correction of CFTR in both airway and GI tissues with no off-target effects above background. Our studies demonstrate that systemic gene editing is possible, and more specifically that intravenous delivery of PNA NPs designed to correct CF-causing mutations is a viable option to ameliorate CF in multiple affected organs.

Calcium-modulated chloride pathways contribute to chloride flux in murine cystic fibrosis-affected macrophages.

Cystic fibrosis (CF), a common lethal inherited disorder defined by ion transport abnormalities, chronic infection, and robust inflammation, is the result of mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein, a cAMP-activated chloride (Cl-) channel. Macrophages are reported to have impaired activity in CF. Previous studies suggest that Cl- transport is important for macrophage function; therefore, impaired Cl- secretion may underlie CF macrophage dysfunction. To determine whether alterations in Cl- transport exist in CF macrophages, Cl- efflux was measured using N-[ethoxycarbonylmethyl]- 6-methoxy-quinolinium bromide (MQAE), a fluorescent indicator dye. The contribution of CFTR was assessed by calculating Cl- flux in the presence and absence of cftr(inh)-172. The contribution of calcium (Ca(2+))-modulated Cl- pathways was assessed by examining Cl- flux with varied extracellular Ca(2+) concentrations or after treatment with carbachol or thapsigargin, agents that increase intracellular Ca(2+) levels. Our data demonstrate that CFTR contributed to Cl- efflux only in WT macrophages, while Ca(2+)-mediated pathways contributed to Cl- transport in CF and WT macrophages. Furthermore, CF macrophages demonstrated augmented Cl- efflux with increases in extracellular Ca(2+). Taken together, this suggests that Ca(2+)-mediated Cl- pathways are enhanced in CF macrophages compared with WT macrophages.

Calcium-pump inhibitors induce functional surface expression of Delta F508-CFTR protein in cystic fibrosis epithelial cells.

The most common mutation in cystic fibrosis, Delta F508, results in a cystic fibrosis transmembrane conductance regulator (CFTR) protein that is retained in the endoplasmic reticulum (ER). Retention is dependent upon chaperone proteins, many of which require Ca(++) for optimal activity. Interfering with chaperone activity by depleting ER Ca(++) stores might allow functional Delta F508-CFTR to reach the cell surface. We exposed several cystic fibrosis cell lines to the ER Ca(++) pump inhibitor thapsigargin and evaluated surface expression of Delta F508-CFTR. Treatment released ER-retained Delta F508-CFTR to the plasma membrane, where it functioned effectively as a Cl(-) channel. Treatment with aerosolized calcium-pump inhibitors reversed the nasal epithelial potential defect observed in a mouse model of Delta F508-CFTR expression. Thus, ER calcium-pump inhibitors represent a potential target for correcting the cystic fibrosis defect.

Emerging technologies for cystic fibrosis transmembrane conductance regulator restoration in all people with CF.

Although effective cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapy has the potential to change the lives of many patients with cystic fibrosis (CF), it is unlikely that these drugs will be a game changing therapy for all. There are about 10% of patients with CF who don't produce a mutant protein tomodulate, potentiate, or optimize and for these patients such therapies are unlikely to be of significant benefit. There is a need to develop new therapeutic approaches that can work for this patient population and can advance CF therapies. These new therapies will be genetic-based therapies and each approach will result in functional CFTR protein inpreviously affected CF cells. In this review we will examine the potential of RNA therapies, gene transfer therapies, and gene editing therapies for the treatment of CF as well as the challenges that will need to be facedas we harness the power of these emerging therapies towards a one-time cure.

Nanoparticles for delivery of agents to fetal lungs.

Fetal treatment of congenital lung disease, such as cystic fibrosis, surfactant protein syndromes, and congenital diaphragmatic hernia, has been made possible by improvements in prenatal diagnostic and interventional technology. Delivery of therapeutic agents to fetal lungs in nanoparticles improves cellular uptake. The efficacy and safety of nanoparticle-based fetal lung therapy depends on targeting of necessary cell populations. This study aimed to determine the relative distribution of nanoparticles of a variety of compositions and sizes in the lungs of fetal mice delivered through intravenous and intra-amniotic routes. Intravenous delivery of particles was more effective than intra-amniotic delivery for epithelial, endothelial and hematopoietic cells in the fetal lung. The most effective targeting of lung tissue was with 250nm Poly-Amine-co-Ester (PACE) particles accumulating in 50% and 44% of epithelial and endothelial cells. This study demonstrated that route of delivery and particle composition impacts relative cellular uptake in fetal lung, which will inform future studies in particle-based fetal therapy.

Understanding the Host in the Management of Pneumonia. An Official American Thoracic Society Workshop Report.

Pneumonia causes a significant burden of disease worldwide. Although all populations are at risk of pneumonia, those at extremes of age and those with immunosuppressive disorders, underlying respiratory disease, and critical illness are particularly vulnerable. Although clinical practice guidelines addressing the management and treatment of pneumonia exist, few of the supporting studies focus on the crucial contributions of the host in pneumonia pathogenesis and recovery. Such essential considerations include the host risk factors that lead to susceptibility to lung infections; biomarkers reflecting the host response and the means to pursue host-directed pneumonia therapy; systemic effects of pneumonia on the host; and long-term health outcomes after pneumonia. To address these gaps, the Pneumonia Working Group of the Assembly on Pulmonary Infection and Tuberculosis led a workshop held at the American Thoracic Society meeting in May 2018 with overarching objectives to foster attention, stimulate research, and promote funding for short-term and long-term investigations into the host contributions to pneumonia. The workshop involved participants from various disciplines with expertise in lung infection, pneumonia, sepsis, immunocompromised patients, translational biology, data science, genomics, systems biology, and clinical trials. This workshop report summarizes the presentations and discussions and important recommendations for future clinical pneumonia studies. These recommendations include establishing consensus disease and outcome definitions, improved phenotyping, development of clinical study networks, standardized data and biospecimen collection and protocols, and development of innovative trial designs.