Loss of Paneth cells dysregulates gut ILC subsets and enhances weight gain response to high fat diet in a mouse model.

Obesity has been associated with dysbiosis, but innate mechanisms linking intestinal epithelial cell subsets and obesity remain poorly understood. Using mice lacking Paneth cells (Sox9 ΔIEC mice), small intestinal epithelial cells specialized in the production of antimicrobial products and cytokines, we show that dysbiosis alone does not induce obesity or metabolic disorders. Loss of Paneth cells reduced ILC3 and increased ILC2 numbers in the intestinal lamina propria. High-fat diet (HFD) induced higher weight gain and more severe metabolic disorders in Sox9 ΔIEC mice. Further, HFD enhances the number of ILC1 in the intestinal lamina propria of Sox9 ΔIEC mice and increases intestinal permeability and the accumulation of immune cells (inflammatory macrophages and T cells, and B cells) in abdominal fat tissues of obese Sox9 ΔIEC . Transplantation of fecal materials from Sox9 ΔIEC mice in germ-free mice before HFD further confirmed the regulatory role of Paneth cells for gut ILC subsets and the development of obesity.

Single-Stranded DNA with Internal Base Modifications Mediates Highly Efficient Gene Insertion in Primary Cells.

Single-stranded DNA (ssDNA) templates along with Cas9 have been used for gene insertion but suffer from low efficiency. Here, we show that ssDNA with chemical modifications in 10-17% of internal bases (eDNA) is compatible with the homologous recombination machinery. Moreover, eDNA templates improve gene insertion by 2-3 fold compared to unmodified and end-modified ssDNA in airway basal stem cells (ABCs), hematopoietic stem and progenitor cells (HSPCs), T-cells and endothelial cells. Over 50% of alleles showed gene insertion in three clinically relevant loci (CFTR, HBB, and CCR5) in ABCs using eDNA and up to 70% of alleles showed gene insertion in the HBB locus in HSPCs. This level of correction is therapeutically relevant and is comparable to adeno-associated virus-based templates. Knocking out TREX1 nuclease improved gene insertion using unmodified ssDNA but not eDNA suggesting that chemical modifications inhibit TREX1. This approach can be used for therapeutic applications and biological modeling.

Influenza virus reduces ubiquitin E3 ligase MARCH10 expression to decrease ciliary beat frequency.

Respiratory viruses, such as influenza, decrease airway cilia function and expression, which leads to reduced mucociliary clearance and inhibited overall immune defense. Ubiquitination is a posttranslational modification using E3 ligases, which plays a role in the assembly and disassembly of cilia. We examined the role of membrane-associated RING-CH (MARCH) family of E3 ligases during influenza infection and determined that MARCH10, specifically expressed in ciliated epithelial cells, is significantly decreased during influenza infection in mice, human lung epithelial cells, and human lung tissue. Cellular depletion of MARCH10 in differentiated human bronchial epithelial cells (HBECs) using CRISPR/Cas9 showed a decrease in ciliary beat frequency. Furthermore, MARCH10 cellular knockdown in combination with influenza infection selectively decreased immunoreactive levels of the ciliary component, dynein axonemal intermediate chain 1. Cellular overexpression of MARCH10 significantly decreased influenza hemagglutinin protein levels in the differentiated HBECs and knockdown of MARCH10 increased IL-1ß cytokine expression, whereas overexpression had the reciprocal effect. These findings suggest that MARCH10 may have a protective role in airway pulmonary host defense and innate immunity during influenza infection.

Architecture and matrix assembly determinants of Bordetella pertussis biofilms on primary human airway epithelium.

Traditionally, whooping cough or pertussis caused by the obligate human pathogen Bordetella pertussis (Bp) is described as an acute disease with severe symptoms. However, many individuals who contract pertussis are either asymptomatic or show very mild symptoms and yet can serve as carriers and sources of bacterial transmission. Biofilms are an important survival mechanism for bacteria in human infections and disease. However, bacterial determinants that drive biofilm formation in humans are ill-defined. In the current study, we show that Bp infection of well-differentiated primary human bronchial epithelial cells leads to formation of bacterial aggregates, clusters, and highly structured biofilms which are colocalized with cilia. These findings mimic observations from pathological analyses of tissues from pertussis patients. Distinct arrangements (mono-, bi-, and tri-partite) of the polysaccharide Bps, extracellular DNA, and bacterial cells were visualized, suggesting complex heterogeneity in bacteria-matrix interactions. Analyses of mutant biofilms revealed positive roles in matrix production, cell cluster formation, and biofilm maturity for three critical Bp virulence factors: Bps, filamentous hemagglutinin, and adenylate cyclase toxin. Adherence assays identified Bps as a new Bp adhesin for primary human airway cells. Taken together, our results demonstrate the multi-factorial nature of the biofilm extracellular matrix and biofilm development process under conditions mimicking the human respiratory tract and highlight the importance of model systems resembling the natural host environment to investigate pathogenesis and potential therapeutic strategies.

5-methylcytosine (m5C) RNA modification controls the innate immune response to virus infection by regulating type I interferons.

5-methylcytosine (m5C) is one of the most prevalent modifications of RNA, playing important roles in RNA metabolism, nuclear export, and translation. However, the potential role of RNA m5C methylation in innate immunity remains elusive. Here, we show that depletion of NSUN2, an m5C methyltransferase, significantly inhibits the replication and gene expression of a wide range of RNA and DNA viruses. Notably, we found that this antiviral effect is largely driven by an enhanced type I interferon (IFN) response. The antiviral signaling pathway is dependent on the cytosolic RNA sensor RIG-I but not MDA5. Transcriptome-wide mapping of m5C following NSUN2 depletion in human A549 cells revealed a marked reduction in the m5C methylation of several abundant noncoding RNAs (ncRNAs). However, m5C methylation of viral RNA was not noticeably altered by NSUN2 depletion. In NSUN2-depleted cells, the host RNA polymerase (Pol) III transcribed ncRNAs, in particular RPPH1 and 7SL RNAs, were substantially up-regulated, leading to an increase of unshielded 7SL RNA in cytoplasm, which served as a direct ligand for the RIG-I-mediated IFN response. In NSUN2-depleted cells, inhibition of Pol III transcription or silencing of RPPH1 and 7SL RNA dampened IFN signaling, partially rescuing viral replication and gene expression. Finally, depletion of NSUN2 in an ex vivo human lung model and a mouse model inhibits viral replication and reduces pathogenesis, which is accompanied by enhanced type I IFN responses. Collectively, our data demonstrate that RNA m5C methylation controls antiviral innate immunity through modulating the m5C methylome of ncRNAs and their expression.

Assemblies of JAG1 and JAG2 determine tracheobronchial cell fate in mucosecretory lung disease.

Mucosecretory lung disease compromises airway epithelial function and is characterized by goblet cell hyperplasia and ciliated cell hypoplasia. Goblet and ciliated cell types are derived from tracheobronchial stem/progenitor cells via a Notch-dependent mechanism. Although specific arrays of Notch receptors regulate cell fate determination, the function of the ligands Jagged1 (JAG1) and JAG2 is unclear. This study examined JAG1 and JAG2 function using human air-liquid-interface cultures that were treated with γ-secretase complex (GSC) inhibitors, neutralizing peptides/antibodies, or WNT/ß-catenin pathway antagonists/agonists. These experiments revealed that JAG1 and JAG2 regulated cell fate determination in the tracheobronchial epithelium; however, their roles did not adhere to simple necessity and sufficiency rules. Biochemical studies indicated that JAG1 and JAG2 underwent posttranslational modifications that resulted in generation of a JAG1 C-terminal peptide and regulated the abundance of full-length JAG2 on the cell surface. GSC and glycogen synthase kinase 3 were implicated in these posttranslational events, but WNT agonist/antagonist studies and RNA-Seq indicated a WNT-independent mechanism. Collectively, these data suggest that posttranslational modifications create distinct assemblies of JAG1 and JAG2, which regulate Notch signal strength and determine the fate of tracheobronchial stem/progenitor cells.

Caspase-4/11 exacerbates disease severity in SARS-CoV-2 infection by promoting inflammation and immunothrombosis.

Severe acute respiratory syndrome coronavirus 2 (SARS­CoV-2) is a worldwide health concern, and new treatment strategies are needed. Targeting inflammatory innate immunity pathways holds therapeutic promise, but effective molecular targets remain elusive. Here, we show that human caspase-4 (CASP4) and its mouse homolog, caspase-11 (CASP11), are up-regulated in SARS­CoV-2 infections and that CASP4 expression correlates with severity of SARS­CoV-2 infection in humans. SARS­CoV-2­infected Casp11−/− mice were protected from severe weight loss and lung pathology, including blood vessel damage, compared to wild-type (WT) mice and mice lacking the caspase downstream effector gasdermin-D (Gsdmd−/−). Notably, viral titers were similar regardless of CASP11 knockout. Global transcriptomics of SARS­CoV-2­infected WT, Casp11−/−, and Gsdmd−/− lungs identified restrained expression of inflammatory molecules and altered neutrophil gene signatures in Casp11−/− mice. We confirmed that protein levels of inflammatory mediators interleukin (IL)-1ß, IL-6, and CXCL1, as well as neutrophil functions, were reduced in Casp11−/− lungs. Additionally, Casp11−/− lungs accumulated less von Willebrand factor, a marker for endothelial damage, but expressed more Kruppel-Like Factor 2, a transcription factor that maintains vascular integrity. Overall, our results demonstrate that CASP4/11 promotes detrimental SARS­CoV-2­induced inflammation and coagulopathy, largely independently of GSDMD, identifying CASP4/11 as a promising drug target for treatment and prevention of severe COVID-19.

Targeting the EGFR-ERK axis using the compatible solute ectoine to stabilize CFTR mutant F508del.

Mutations in the CFTR gene lead to cystic fibrosis, a genetic disease associated with chronic infection and inflammation and ultimately respiratory failure. The most common CF-causing mutation is F508del and CFTR modulators (correctors and potentiators) are being developed to rescue its trafficking and activity defects. However, there are currently no modulators that stabilize the rescued membrane F508del-CFTR which is endocytosed and quickly degraded resulting in a shorter half-life than wild-type (WT). We previously reported that the extracellular signal-regulated kinase (ERK) MAPK pathway is involved in CFTR degradation upon cigarette smoke exposure. Interestingly, we found that ERK phosphorylation was increased in CF human bronchial epithelial (HBE) cells (CF-HBE41o- and primary CF-HBE) compared to non-CF controls, and this was likely due to signaling by the epidermal growth factor receptor (EGFR). EGFR can be activated by several ligands, and we provide evidence that amphiregulin (AREG) is important for activating this signaling axis in CF. The natural osmolyte ectoine stabilizes membrane macromolecules. We show that ectoine decreases ERK phosphorylation, increases the half-life of rescued CFTR, and increases CFTR-mediated chloride transport in combination with the CFTR corrector VX-661. Additionally, ectoine reduces production of AREG and interleukin-8 by CF primary bronchial epithelial cells. In conclusion, EGFR-ERK signaling negatively regulates CFTR and is hyperactive in CF, and targeting this axis with ectoine may prove beneficial for CF patients.

Differential gene expression of 3D primary human airway cultures exposed to cigarette smoke and electronic nicotine delivery system (ENDS) preparations.

BACKGROUND: Acute exposure to cigarette smoke alters gene expression in several biological pathways such as apoptosis, immune response, tumorigenesis and stress response, among others. However, the effects of electronic nicotine delivery systems (ENDS) on early changes in gene expression is relatively unknown. The objective of this study was to evaluate the early toxicogenomic changes using a fully-differentiated primary normal human bronchial epithelial (NHBE) culture model after an acute exposure to cigarette and ENDS preparations. RESULTS: RNA sequencing and pathway enrichment analysis identified time and dose dependent changes in gene expression and several canonical pathways when exposed to cigarette preparations compared to vehicle control, including oxidative stress, xenobiotic metabolism, SPINK1 general cancer pathways and mucociliary clearance. No changes were observed with ENDS preparations containing up to 28 µg/mL nicotine. Full model hierarchical clustering revealed that ENDS preparations were similar to vehicle control. CONCLUSION: This study revealed that while an acute exposure to cigarette preparations significantly and differentially regulated many genes and canonical pathways, ENDS preparations containing the same concentration of nicotine had very little effect on gene expression in fully-differentiated primary NHBE cultures.

CFTR Modulators Restore Acidification of Autophago-Lysosomes and Bacterial Clearance in Cystic Fibrosis Macrophages.

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.

Pollutants enhance IgE sensitization in the gut via local alteration of vitamin D-metabolizing enzymes.

Mechanisms linking ingested pollutants to increased incidence of allergy are poorly understood. We report that mice exposed to low doses of cadmium develop higher IgE responses following oral allergen sensitization and more severe allergic symptoms upon allergen challenge. The environmentally relevant doses of this pollutant also induced oxidative/inflammatory responses in the gut of SPF, but not germ-free mice. Interestingly, the increased IgE responses correlated with stimulation of the vitamin D3-metabolizing enzymes CYP27B1 and CYP24A1 in the gut and increased luminal levels of oxidized vitamin D3 metabolites that are not ligands of the vitamin D receptor. Inhibition of CYP27B1 and CYP24A1 via oral administration of pharmacological inhibitors reduced IgE responses induced in mice orally exposed to cadmium. Our findings identify local alteration of vitamin D signaling as a new mechanism for induction of IgE responses by environmental pollutants. They also identify vitamin D3-metabolizing enzymes as therapeutic targets for the treatment of allergy.

Transcriptomic Response of Primary Human Bronchial Cells to Repeated Exposures of Cigarette and ENDS Preparations.

Cigarette smoke deregulates several biological pathways by modulating gene expression in airway epithelial cells and altering the physiology of the airway epithelium. The effects of repeated exposures of electronic cigarette delivery systems (ENDS) on gene expression in airway epithelium are relatively unknown. In order to assess the effect of repeated exposures of ENDS, primary normal human bronchial epithelial (NHBE) cells grown at air-liquid interface (ALI) were exposed to cigarette and ENDS preparations daily for 10 days. Cigarette smoke preparations significantly altered gene expression in a dose-dependent manner compared to vehicle control, including genes linked to oxidative stress, xenobiotic metabolism, cancer pathways, epithelial-mesenchymal transition, fatty acid metabolism, degradation of collagen and extracellular matrix, O-glycosylation, and chemokines/cytokines, which are known pathways found to be altered in smokers. Conversely, ENDS preparations had minimal effect on transcriptional pathways. This study revealed that a sub-chronic exposure of primary NHBE cultures to cigarette and ENDS preparations differentially regulated genes and canonical pathways, with minimal effect observed with ENDS preparations compared to cigarette preparations. This study also demonstrates the versatility of primary NHBE cultures at ALI to evaluate repeat-dose exposures of tobacco products.

Inhibition of elastase enhances the adjuvanticity of alum and promotes anti-SARS-CoV-2 systemic and mucosal immunity.

Alum, used as an adjuvant in injected vaccines, promotes T helper 2 (Th2) and serum antibody (Ab) responses. However, it fails to induce secretory immunoglobulin (Ig) A (SIgA) in mucosal tissues and is poor in inducing Th1 and cell-mediated immunity. Alum stimulates interleukin 1 (IL-1) and the recruitment of myeloid cells, including neutrophils. We investigated whether neutrophil elastase regulates the adjuvanticity of alum, and whether a strategy targeting neutrophil elastase could improve responses to injected vaccines. Mice coadministered a pharmacological inhibitor of elastase, or lacking elastase, developed high-affinity serum IgG and IgA antibodies after immunization with alum-adsorbed protein vaccines, including the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2). These mice also developed broader antigen-specific CD4+ T cell responses, including high Th1 and T follicular helper (Tfh) responses. Interestingly, in the absence of elastase activity, mucosal SIgA responses were induced after systemic immunization with alum as adjuvant. Importantly, lack or suppression of elastase activity enhanced the magnitude of anti-SARS-CoV-2 spike subunit 1 (S1) antibodies, and these antibodies reacted with the same epitopes of spike 1 protein as sera from COVID-19 patients. Therefore, suppression of neutrophil elastase could represent an attractive strategy for improving the efficacy of alum-based injected vaccines for the induction of broad immunity, including mucosal immunity.

Elevated Expression of MiR-17 in Microglia of Alzheimer's Disease Patients Abrogates Autophagy-Mediated Amyloid-ß Degradation.

Autophagy is a proposed route of amyloid-ß (Aß) clearance by microglia that is halted in Alzheimer's Disease (AD), though mechanisms underlying this dysfunction remain elusive. Here, primary microglia from adult AD (5xFAD) mice were utilized to demonstrate that 5xFAD microglia fail to degrade Aß and express low levels of autophagy cargo receptor NBR1. In 5xFAD mouse brains, we show for the first time that AD microglia express elevated levels of microRNA cluster Mirc1/Mir17-92a, which is known to downregulate autophagy proteins. By in situ hybridization in post-mortem AD human tissue sections, we observed that the Mirc1/Mir17-92a cluster member miR-17 is also elevated in human AD microglia, specifically in the vicinity of Aß deposits, compared to non-disease controls. We show that NBR1 expression is negatively correlated with expression of miR-17 in human AD microglia via immunohistopathologic staining in human AD brain tissue sections. We demonstrate in healthy microglia that autophagy cargo receptor NBR1 is required for Aß degradation. Inhibiting elevated miR-17 in 5xFAD mouse microglia improves Aß degradation, autophagy, and NBR1 puncta formation in vitro and improves NBR1 expression in vivo. These findings offer a mechanism behind dysfunctional autophagy in AD microglia which may be useful for therapeutic interventions aiming to improve autophagy function in AD.

Association of cystic fibrosis transmembrane conductance regulator with epithelial sodium channel subunits carrying Liddle's syndrome mutations.

The association of the cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC) in the pathophysiology of cystic fibrosis (CF) is controversial. Previously, we demonstrated a close physical association between wild-type (WT) CFTR and WT ENaC. We have also shown that the F508del CFTR fails to associate with ENaC unless the mutant protein is rescued pharmacologically or by low temperature. In this study, we present the evidence for a direct physical association between WT CFTR and ENaC subunits carrying Liddle's syndrome mutations. We show that all three ENaC subunits bearing Liddle's syndrome mutations (both point mutations and the complete truncation of the carboxy terminus), could be coimmunoprecipitated with WT CFTR. The biochemical studies were complemented by fluorescence lifetime imaging microscopy (FLIM), a distance-dependent approach that monitors protein-protein interactions between fluorescently labeled molecules. Our measurements revealed significantly increased fluorescence resonance energy transfer between CFTR and all tested ENaC combinations as compared with controls (ECFP and EYFP cotransfected cells). Our findings are consistent with the notion that CFTR and ENaC are within reach of each other even in the setting of Liddle's syndrome mutations, suggestive of a direct intermolecular interaction between these two proteins.

SARS-CoV-2 may hijack GPCR signaling pathways to dysregulate lung ion and fluid transport.

The tropism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a virus responsible for the ongoing coronavirus disease 2019 (COVID-19) pandemic, toward the host cells is determined, at least in part, by the expression and distribution of its cell surface receptor, angiotensin-converting enzyme 2 (ACE2). The virus further exploits the host cellular machinery to gain access into the cells; its spike protein is cleaved by a host cell surface transmembrane serine protease 2 (TMPRSS2) shortly after binding ACE2, followed by its proteolytic activation at a furin cleavage site. The virus primarily targets the epithelium of the respiratory tract, which is covered by a tightly regulated airway surface liquid (ASL) layer that serves as a primary defense mechanism against respiratory pathogens. The volume and viscosity of this fluid layer is regulated and maintained by a coordinated function of different transport pathways in the respiratory epithelium. We argue that SARS-CoV-2 may potentially alter evolutionary conserved second-messenger signaling cascades via activation of G protein-coupled receptors (GPCRs) or by directly modulating G protein signaling. Such signaling may in turn adversely modulate transepithelial transport processes, especially those involving cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial Na+ channel (ENaC), thereby shifting the delicate balance between anion secretion and sodium absorption, which controls homeostasis of this fluid layer. As a result, activation of the secretory pathways including CFTR-mediated Cl- transport may overwhelm the absorptive pathways, such as ENaC-dependent Na+ uptake, and initiate a pathophysiological cascade leading to lung edema, one of the most serious and potentially deadly clinical manifestations of COVID-19.

Airway epithelial stem cell chimerism in cystic fibrosis lung transplant recipients.

BACKGROUND: The conducting airway epithelium is repaired by tissue specific stem cells (TSC). In response to mild/moderate injury, each TSC repairs a discrete area of the epithelium. In contrast, severe epithelial injury stimulates TSC migration and expands the stem cell's reparative domain. Lung transplantation (LTx) can cause a moderate/severe airway injury and the remodeled airway contains a chimeric mixture of donor and recipient cells. These studies supported the hypothesis, LTx stimulates TSC migration resulting in epithelial chimerism. We tested this hypothesis in cystic fibrosis (CF) LTx patients. METHODS: Airway mucosal injury was quantified using bronchoscopic imaging and a novel grading system. Bronchial brushing was used to recover TSC from 10 sites in the recipient and allograft airways. TSC chimerism was quantified by short tandem repeat analysis. TSC self-renewal and differentiation potential were assayed using the clone forming cell frequency and air-liquid-interface methods. Electrophysiology was used to determine if TSC chimerism altered epithelial ion channel activity. RESULTS: LTx caused a mild to moderate airway mucosal injury. Donor and recipient TSC were identified in 91% of anastomotic sites and 93% of bronchial airways. TSC chimerism did not alter stem cell self-renewal or differentiation potential. The frequency of recipient TSC was proportional to CF Transmembrane Conductance Regulator (CFTR)-dependent ion channel activity and 33% of allograft regions were at risk for abnormal CFTR activity. CONCLUSIONS: LTx in CF patients stimulates bidirectional TSC migration across the anastomoses. TSC chimerism may alter ion homeostasis and compromise the host defense capability of the allograft airway epithelium.

Cigarette smoke preparations, not electronic nicotine delivery system preparations, induce features of lung disease in a 3D lung repeat-dose model.

Cigarette smoking is a risk factor for several lung diseases, including chronic obstructive pulmonary disease, cardiovascular disease, and lung cancer. The potential health effects of chronic use of electronic nicotine delivery systems (ENDS) is unclear. This study utilized fully differentiated primary normal human bronchial epithelial (NHBE) cultures in a repeat-dose exposure to evaluate and compare the effect of combustible cigarette and ENDS preparations. We show that 1-h daily exposure of NHBE cultures over a 10-day period to combustible cigarette whole smoke-conditioned media (WS-CM) increased expression of oxidative stress markers, cell proliferation, airway remodeling, and cellular transformation markers and decreased mucociliary function including ion channel function and airway surface liquid. Conversely, aerosol conditioned media (ACM) from ENDS with similar nicotine concentration (equivalent-nicotine units) as WS-CM and nicotine alone had no effect on those parameters. In conclusion, primary NHBE cultures in a repeat-dose exposure system represent a good model to assess the features of lung disease. This study also reveals that cigarette and ENDS preparations differentially elicit several key endpoints, some of which are potential biomarkers for lung cancer or chronic obstructive pulmonary disease (COPD).

Cyclic Peptidyl Inhibitors against CAL/CFTR Interaction for Treatment of Cystic Fibrosis.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, encoding for a chloride ion channel. Membrane expression of CFTR is negatively regulated by CFTR-associated ligand (CAL). We previously showed that inhibition of the CFTR/CAL interaction with a cell-permeable peptide improves the function of rescued F508del-CFTR. In this study, optimization of the peptidyl inhibitor yielded PGD97, which exhibits a KD value of 6 nM for the CAL PDZ domain, ≥ 130-fold selectivity over closely related PDZ domains, and a serum t1/2 of >24 h. In patient-derived F508del homozygous cells, PGD97 (100 nM) increased short-circuit currents by ∼3-fold and further potentiated the therapeutic effects of small-molecule correctors (e.g., VX-661) by ∼2-fold (with an EC50 of ∼10 nM). Our results suggest that PGD97 may be used as a novel treatment for CF, either as a single agent or in combination with small-molecule correctors/potentiators.