FASEB J. 2022 May;36 Suppl 1. doi: 10.1096/fasebj.2022.36.S1.R6263.

ABSTRACT

The Na/K ATPase localizes within the basolateral membrane of most epithelia, where Na/K ATPase activity mediates Na+ absorption. The Na/K ATPase requires its alpha subunit for ion transport and its beta subunit for proper trafficking to the plasma membrane. The non-essential gamma subunit of the Na/K ATPase modifies Na/K ATPase activity. Datasets within the NIH Gene Expression Omnibus indicate that FXYD3, a Na/K ATPase gamma subunit, is highly expressed by human airway epithelia. However, how FXYD3 affects airway epithelial function remains unstudied. In addition to binding the Na/K ATPase, FXYD3 can bind and modify H/K ATPases in heterologous expression systems. The H/K ATPase ATP12A acidifies human airway surface liquid. Therefore, we evaluated the role of FXYD3 in both acid secretion and Na+ absorption across human airway epithelia. Single cell RNA sequencing revealed high FXYD3 expression for all the airway cell types, and immunocytochemistry revealed FXYD3 expression within the basolateral membrane of airway epithelia. Consistent with basolateral FXYD3 localization, the airway surface liquid pH, established in part by apical H/K ATPase, was unaffected by siRNA-mediated knockdown of FXYD3. FXYD3 knockdown decreased amiloride-sensitive short-circuit currents (~20% in NaCl and ~40% in NaGluconate solutions), a finding that is consistent with FXYD3 increasing Na/K ATPase activity. Consequently, fluid absorption across airway epithelia was reduced by ~20% with FXYD3 knockdown. FXYD3 may be required to efficiently return the airway surface liquid to homeostatic volumes after fluid secretion into the proximal airways.

PMID:35556371 | DOI:10.1096/fasebj.2022.36.S1.R6263

FASEB J. 2022 May;36 Suppl 1. doi: 10.1096/fasebj.2022.36.S1.R3616.

ABSTRACT

Pseudomonas aeruginosa is an opportunistic human pathogen that causes nosocomial, acute, and chronic infections in patients with conditions such as cystic fibrosis, severe burns, and cancer. P. aeruginosa is known for its persistence in infections due largely to its numerous means of antimicrobic resistance, one of which is its ability to form biofilms. This organism has been placed on the World Health Organization list of Priority Pathogens for Research and Development of New Antibiotics at the critical level for many reasons, that includes its high mortality, healthcare burden, prevalence of resistance, and treatability. P. aeruginosa is resistant to many antibiotics; however, >70% of P. aeruginosa are susceptible to ciprofloxacin. For these reasons, the purpose of the current study was to establish antimicrobic levels of ciprofloxacin effective against P. aeruginosa. To accomplish this purpose, antimicrobic levels of bacteria in biofilms and those not associated with a biofilm (planktonic) need separate methods of testing. For planktonic bacteria, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) are performed, whereas other methods are used for biofilm-associated bacteria. To initiate inhibition studies, a model system was identified of ciprofloxacin effect on standardized strains of P. aeruginosa (ATCC strains 10145, 27853, 1744, CRM-9027, 9721). These strains were used to establish ciprofloxacin effect, in terms of MIC and MBC levels, on planktonic bacteria. Results indicate that MIC and MBC levels of these strains are in the same range as reported values of P. aeruginosa. These results establish ciprofloxacin efficacy of growth inhibition for each ATCC strain of planktonic P. aeruginosa and will serve as baseline results for future studies, such as effect on biofilm-associated bacteria and combination antimicrobic treatments.

PMID:35555795 | DOI:10.1096/fasebj.2022.36.S1.R3616

FASEB J. 2022 May;36 Suppl 1. doi: 10.1096/fasebj.2022.36.S1.R6106.

ABSTRACT

OBJECTIVE: Chloride and bicarbonate transport across the apical membrane via CFTR is critical for homeostasis of airway surface liquid and mucociliary clearance. Cystic fibrosis (CF) is caused by mutations in the CFTR gene resulting in mucus buildup, bacterial colonization, inflammation and respiratory insufficiency. F508del and nonsense mutations are the most common mutations characterized by defective protein processing and trafficking (F508del) or early termination of CFTR synthesis (G542X, R1162X). A combination of CFTR modulators (VX445/661/770; VX) has largely improved CFTR function in patients with F508del but is ineffective in nonsense mutations. We have shown that select amino acids (SAA) can modulate trafficking and activity of CFTR and alternative anion-secretory pathways, and thus could improve clinical outcomes in CF patients.

HYPOTHESIS: SAA can stimulate anion secretion in primary HBEC with F508del/F508del, F508del/R1162X and G542X/G542X mutation.

METHODS: Transepithelial short-circuit current (Isc ) was measured in differentiated HBEC treated with DMSO or VX for 24h and bathed in vehicle or SAA in Ussing chambers. After blocking ENaC with benzamil, benzamil-insensitive (anion) Isc was either continuously recorded for 90min, or sequentially blocked with CFTRinh172, CaCCinhA01 and bumetanide in 15min intervals. Isotope (36 Cl) flux studies and whole-cell patch clamp technique were used to confirm chloride movements, and CFTR and alternative anion-secretory channel expression was analyzed by immunofluorescence.

RESULTS: Anion Isc increased in all mutations and was more sustained when exposed to SAA compared to vehicle (F508del: 15.4±0.5 vs 1.4±0.2; R1162X: 19.4±0.3 vs 2.3±0.3; G542X: 17.2±0.5 vs 1.6±0.2 µA.cm-2 ; P<0.001). SAA recovered 48.7, 57.3 and 40.9% of normal Isc (21.9±0.5 µA.cm-2 ). Treatment with VX increased anion Isc in F508del and R1162X (11.4±0.7 and 5.7±0.4 µA.cm-2 ; P<0.001) but not in G542X (1.7±0.2 µA.cm-2 ). In F508del, the CFTR response increased from 20.7% (non-treated cells) to 32.9 and 85.5% of the anion Isc when treated with SAA or VX, and in R542X from 3.7% to 46.3 and 16.0%, respectively. Both, SAA and VX did not affect CaCC, but SAA increased the bumetanide-sensitive Isc from 31.0 to 50.5% in F508del, and from 33.3 to 56.3% in R1162X, while VX reduced the bumetanide response to 5.4 and 4.5%. Isotope flux studies showed that SAA (-0.21±0.08 μEq.h-1 .cm-2 ) and VX (-0.19±0.02 μEq.h-1 .cm-2 ) increased net Cl- secretion in F508del when compared to vehicle (-0.03±0.01 μEq.h-1 .cm-2 ; P<0.05). SAA, but not VX increased Cl- secretion in G542X (-0.21 0.07 vs -0.08±0.02 μEq.h-1 .cm-2 ). In patch clamp recordings, SAA and VX resulted in a significant reduction in CFTRinh172 whole cell current in F508del. Immunofluorescence imaging demonstrated increased CFTR and alternative anion-secretory channel expression along the apical membrane in SAA cells while VX showed increased expression of only CFTR.

CONCLUSIONS: These studies demonstrate that SAA stimulates anion current and increases Cl- secretion via CFTR and alternative secretory pathways in different mutations, and could be beneficial in nonsense mutations but needs further investigation.

PMID:35555194 | DOI:10.1096/fasebj.2022.36.S1.R6106

FASEB J. 2022 May;36 Suppl 1. doi: 10.1096/fasebj.2022.36.S1.R2630.

ABSTRACT

Invasive candidiasis is one of the most common causes of hospital-acquired blood infections, with the most common cause being Candida albicans. These bloodstream infections often lead to long hospital stays and have a high mortality rate. Once C. albicansenters the bloodstream it can disseminate and invade many organs of the body, including the lungs, where it can inhibit function and cause further complications. Furthermore, C. albicansis commonly isolated from the lungs of cystic fibrosis patients and often causes coinfection in patients with severe COVID-19. Current antifungal drugs exhibit high toxicity, poor water solubility, and are ineffective against some strains of C. albicans due to antifungal resistance, creating an urgent need for new antifungal therapeutics. Here, we have developed a customizable in vitroplatform to study the interaction between rat lung microvascular endothelial cells (ECs), C. albicansSC5314, and novel liposomes encapsulating the antifungal drug anidulafungin. Liposome formulations composed of hydrogenated soy phosphatidylcholine, phosphatidylglycerol, cholesterol, and α-tocopherol were fabricated containing three different % wt/wt concentrations of anidulafungin (0.31, 1.55, and 3.10, or 1x, 5x and 10x, respectively). The minimum inhibitory concentrations (MICs) of the different liposome formulations for C. albicans were determined using a microdilution assay. After 24 hours of exposure, the MICs were found to be 12.50, 6.25, and 1.56 μg/mL for 1x, 5x, and 10x liposome formulations, respectively. Cell Counting Kit 8 was used to determine EC viability after 24-hour treatment with the different liposome formulations. The 1x and 5x anidulafungin liposome formulations showed no significant decrease in viability up to the maximum liposome concentration tested (50 μg/mL), with the 10x formulation causing a slight but significant decrease in viability. The 1x and 5x anidulafungin liposomes were then assessed at 6.25 and 12.5 μg/mL in a co-culture setting. C. albicans were seeded onto established EC monolayers and were allowed to form hyphae, which is the invasive form. After the two hours, the co-cultures were treated with the antifungal liposomes. Treatment with the liposomes reduced hyphae formation and prevented C. albicansovergrowth within four hours of exposure, with the 5x anidulafungin liposome at 12.5 μg/mL showing the strongest change. These data suggest that these novel antifungal liposomes have the potential to treat pulmonary candidiasis without the toxic side effects associated with current antifungal drugs.

PMID:35555184 | DOI:10.1096/fasebj.2022.36.S1.R2630

FASEB J. 2022 May;36 Suppl 1. doi: 10.1096/fasebj.2022.36.S1.R3074.

ABSTRACT

Pseudomonas aeruginosa can cause severe opportunistic pulmonary infections associated with mechanical ventilation and the genetic disease cystic fibrosis. Due to its multidrug resistance, it is a priority pathogen for therapeutic development. Inflammation during such infections, which causes destruction of lung architecture and function, is activated in part through the maturation of IL-1β by the virulence factor LasB. Previous results in mouse models suggest that matrix metalloprotease inhibitor drugs also inhibit LasB, preventing cell death and inflammation caused by caused by P. aeruginosa. We hypothesized that other inhibitors of matrix metalloproteases, including tetracycline-family antibiotics, could be used to target LasB. We evaluated small molecule inhibitors against LasB activation during infections of macrophages, neutrophils, and monocytes and the synergy of these inhibitors with conventional antibiotics and anti-inflammatories. We first optimized the drug concentration by testing the ability of the various drugs to inhibit LasB at various dilutions. Using an effective concentration, we then infected the various cell types with P. aeruginosa and analyzed the impact of the inhibitors on cell death and maturation of IL-1β in infected cells. The results indicate that several tetracycline-family compounds inhibit LasB during P. aeruginosa infections and decrease cell death. This was true for compounds that had no antimicrobial activity yet still inhibited LasB. In addition, the results suggest that the drugs target different cell types. Together, we find that tetracyclines may have therapeutic benefits, even against resistant bacteria, due to their ability to decrease inflammation. This may serve as an effective adjunctive therapy during infection to limit tissue damage and give the immune system and antibiotics more time to respond.

PMID:35554153 | DOI:10.1096/fasebj.2022.36.S1.R3074

FASEB J. 2022 May;36 Suppl 1. doi: 10.1096/fasebj.2022.36.S1.R4758.

ABSTRACT

Pseudomonas aeruginosa is an opportunistic human pathogen that causes nosocomial, acute, and chronic infections in patients with conditions such as cystic fibrosis, severe burns, and cancer. P. aeruginosa is associated with increased patient morbidity, mortality, and healthcare costs and is known for its persistence of infections. P. aeruginosa is resistant to many antibiotics; however, most strains are susceptible to colistin. The World Health Organization has identified antimicrobial resistance as a major threat and Antimicrobial Stewardship Programs (ASP) have been initiated. ASP goals are to improve patient outcomes, optimize patient safety, reduce antimicrobial resistance, and control healthcare costs. Recommendations to reduce antimicrobial resistance include optimizing the use of antimicrobials and preventing the transmission of drug-resistant organisms. Using antimicrobic agents wisely leads to optimization of their use. For these reasons, we sought to adopt a model system for testing minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) levels for standardized strains of P. aeruginosa (ATCC strains 10145, 27853, 1744, CRM-9027, 9721) using standardized methods. This study sought to compare results of standardized analysis that used Mueller-Hinton Broth (MHB), the recommended and standardized growth medium, or tryptic soy broth (TSB), the growth medium used in many labs with published MIC and MBC reports. Given the ASP recommendations of optimizing use of antimicrobial agents, it is important to know the impact of variations in and deviations from standardized MIC and MBC methodologies in reporting of research results relevant to antimicrobic agents. This comparison study tested the assumption that MIC and MBC results are the same regardless of whether MHB or TSB is used as the growth medium. Preliminary results suggest that MIC and MBC values obtained by using TSB or MHB may display some degree of variability.

PMID:35553837 | DOI:10.1096/fasebj.2022.36.S1.R4758

FASEB J. 2022 May;36 Suppl 1. doi: 10.1096/fasebj.2022.36.S1.R5339.

ABSTRACT

BACKGROUND: Small intestinal bicarbonate transport, is critical for epithelial protection, digestion, and absorption. Membrane trafficking and/or recycling of transporters is a critical means to modulate their function, however, the cellular mechanisms that drive these processes in the duodenum remain unclear. The inositol 1,4,5-trisphosphate (IP3 ) receptor binding protein released with IP3 (IRBIT) is an intracellular protein that has been shown to regulate localization of bicarbonate transporters outside of the intestine, yet its role in regulating duodenal bicarbonate secretion is unknown.

AIM: To examine duodenal expression and function of IRBIT in regulating bicarbonate transport.

METHODS: Duodenal endoscopic biopsies from subjects without gross or histological evidence of disease were used directly or cultured using traditional apical-in or flipped apical-out enteroids. mRNA expression was evaluated using qPCR and re-analysis of a duodenal sc-RNAseq dataset. Protein expression and localization was determined by Western blot and confocal immunofluorescence, respectively. In vivo bicarbonate secretion was measured in anesthetized mice by duodenal perfusion and back-titration.

RESULTS: IRBIT is highly expressed in human duodenal biopsies, with protein densitometric analysis showing ≥3.5-fold higher expression than Calu-3 airway submucosal gland cells or HEK293 kidney-derived cells (n=1 each). Analysis of duodenal sc-RNAseq data (1,625 cells) showed ACHYL1 in crypts and villi. Immunofluorescence staining of human duodenal biopsies (n=3) showed IRBIT staining along the crypt-villus axis, primarily localizing in E-cadherin positive epithelial cells, but also present in the lamina propria. In patient-derived duodenal enteroids, IRBIT was expressed in both crypt-like (undifferentiated) and villus-like (differentiated) enteroids (n=3 each), with greater IRBIT mRNA and protein expression in the latter, a pattern similar to SLC26A3 and SLC26A6 chloride/bicarbonate exchangers (n=3 each). In sc-RNAseq analysis, AHCYL1 and SLC26A3 were co-expressed in 38% of all villus cells, compared to only 5% co-expressing AHCYL1 and SLC26A6. CFTR-independent duodenal bicarbonate secretion stimulated by linaclotide (10-7 M) was significantly inhibited by phospholipase C inhibition (U7312, 10-6 M; 91±11%, n=3), which we showed disrupts IRBIT membrane localization, or SLC26A3 inhibition (DRAinh -A250, 10-5 M; 68±8%, n=10). Linaclotide stimulation of apical-out enteroids increased both IRBIT (n=3) and SLC26A3 (n=12) membrane trafficking.

SUMMARY AND CONCLUSIONS: IRBIT is highly expressed in the duodenum and appears to functionally interact with SLC26A3. IRBIT may be an attractive target to modulate small intestinal pH in diseases like cystic fibrosis, where impaired bicarbonate secretion contributes to intestinal disease and malnutrition.

PMID:35553821 | DOI:10.1096/fasebj.2022.36.S1.R5339

FASEB J. 2022 May;36 Suppl 1. doi: 10.1096/fasebj.2022.36.S1.R6019.

ABSTRACT

INTRODUCTION AND AIMS: CFTR, a Cl- channel important in regulating intestinal fluid and electrolyte secretion, is implicated in the pathogenesis of a number of intestinal disorders. We have previously shown that bile acids, acting via the nuclear receptor, farnesoid X receptor (FXR), inhibit colonic epithelial CFTR expression. Dietary phytochemicals have been reported to have the capacity to modulate FXR signalling. Here, we set out to investigate mechanisms underlying FXR regulation of CFTR expression, and the potential for therapeutically targeting the receptor with dietary phytochemicals.

METHODS: Monolayers of T84 colonic epithelial cells were treated with the FXR agonist, GW4064 (5 µM) in the absence or presence of a plant phytochemical, designated here as KFS1 (5 µM). mRNA and protein expression were measured by qPCR and Western blotting. FXR binding to the CFTR promoter was investigated by chromatin immunoprecipitation (ChIP). Electrophysiological studies of T84 cells were conducted in Ussing chambers. Studies on human and murine enteroids were carried out with ethical approval from Johns Hopkins University School of Medicine Institutional Review Board and the Institutional Review Board of the University of California San Diego, respectively.

RESULTS: Treatment of T84 monolayers with GW4064 downregulated CFTR mRNA to 0.51 ± 0.06 fold after 12 hrs (n = 12; p < 0.001) and protein levels to 0.28 ± 0.06 fold after 48 hrs, compared to controls (n = 8; p < 0.001). Studies in Ussing chambers showed that GW4064 treatment for 48 hrs inhibited Cl- secretory responses to the Ca2+ - and cAMP-dependent agonists, carbachol (CCh; 100mM) and forskolin (FSK; 10mM), by 79.9 ± 7.5 % and 74.2 ± 8.9 %, respectively. Transcriptomic analysis of human colonic enteroids revealed FXR to be robustly expressed in secretory (crypt-like) cells and that its activation also induced CFTR downregulation. FXR activation did not alter expression or phosphorylation of the p65 subunit of NF-κB, or inhibit its translocation to the nucleus. ChIP-qPCR analysis appears to show enhanced binding of FXR to the CFTR promoter upon GW4064 treatment. The phytochemical, KFS1 (5 mM; 24hrs), upregulated FXR mRNA and protein expression in T84 cells and enhanced GW4064-induced downregulation of CFTR mRNA by 0.28 ± 0.05 fold (n = 8; p <0.01) and protein by 0.25 ± 0.11 fold (n = 4) after 24 hours. Similarly, KFS1 significantly upregulated FXR mRNA expression in murine colonic epithelial enteroids (2.3 ± 0.2; n = 4; p < 0.01) and enhanced GW4064-induced downregulation of CFTR mRNA by 0.5 ± 0.1 fold (n = 4; p < 0.05) compared to GW4064 alone. Finally, KFS1 enhanced FXR inhibition of agonist-induced Cl- secretory responses across T84 cells mounted in Ussing chambers.

CONCLUSION: FXR regulates colonic epithelial CFTR expression and function by a mechanism which appears to involve direct binding of FXR to the CFTR promoter. By virtue of their ability to upregulate FXR expression, and thereby enhance its antisecretory actions, plant extracts containing KFS1 have excellent potential to be developed as FXR-targeted nutraceuticals for the treatment and prevention of intestinal disease.

PMID:35553806 | DOI:10.1096/fasebj.2022.36.S1.R6019

FASEB J. 2022 May;36 Suppl 1. doi: 10.1096/fasebj.2022.36.S1.R5730.

ABSTRACT

Microvillus Inclusion Disease (MVID) is a lethal congenital diarrheal disease resulting from loss of function mutations in the actin motor myosin VB (MYO5B) that regulates apical traffic. MVID remains without treatment to reverse the severe diarrhea that leads to death. MVID diarrhea results from both increased fluid secretion and malabsorption of ions and carbohydrates in the small intestine. Serum and Glucocorticoid-inducible kinase 1 (SGK1), is a potent regulator of ion transporters including cystic fibrosis transmembrane conductance regulator (CFTR) in the intestine. Since SGK1 upregulates CFTR function in the intestine, we hypothesized that loss of SGK1 could potentially reduce MVID diarrhea by decreasing CFTR fluid secretion. Using CRISPR-Cas 9 approaches, we first generated MYO5B-floxed mice and to generate conditional MYO5B-KO (R26cre ER;MYO5Bf/f ) mice, we cross bred with R26cre ER mice. Tamoxifen-treated R26cre ER;MYO5Bf/f mice resulted in characteristic features of human MVID including severe diarrhea, Microvillus Inclusions (MIs), defective apical traffic, depolarization of proteins, and ion transporters including CFTR, NHE3 and DRA. MYO5B-KO mice also showed increased phosphorylation of SGK1 and PDK1 in the intestine. SGK1-floxed (SGK1f/f )mice were crossed with R26cre ER;MYO5Bf/f mice to generate conditional MYO5B/SGK1 DcKO (R26cre ER;MYO5Bf/f ;SGK1f/f ) upon Tamoxifen induction. Surprisingly, tamoxifen treatment (3 day) of MYO5B/SGK1 DcKO mice resulted in more severe diarrhea compared to MYO5B (R26cre ER;MYO5Bf/f ) mice. Immunoblots of intestinal lysates revealed decreased CFTR, increased alpha- and beta-ENaC, increased phosphorylation of PDK1, Nedd4-2 and pPKCιThr563 in MYO5B/SGK1 DcKO vs. MYO5B ScKO mice. Finally, fecal glucose loss was higher with reduced SGLT1 and GLUT2 by immunoblot in MYO5B/SGK1 DcKO vs MYO5B ScKO mice. We conclude that activation of SGK1 pathway and CFTR contribute to worsening of diarrhea and carbohydrate malabsorption in MVID.

PMID:35553440 | DOI:10.1096/fasebj.2022.36.S1.R5730

FASEB J. 2022 May;36 Suppl 1. doi: 10.1096/fasebj.2022.36.S1.R4485.

ABSTRACT

The corticosteroid hormone, aldosterone, markedly enhances K+ secretion throughout the colon - a mechanism critical to its role in maintaining overall K+ balance. Previous studies from our lab have demonstrated that basolateral Na+ -K+ -2Cl- cotransporter 1 (NKCC1) is transcriptionally up-regulated by aldosterone in the distal colon specifically to support large conductance K+ (BK) channel-mediated K+ secretion. This function is distinct from the more well-established role of NKCC1 in supporting luminal Cl- secretion throughout the gastrointestinal tract. However, considerable segmental variability exists between proximal and distal colonic ion transport processes, especially concerning their regulation by corticosteroids. Although active K+ secretion has also been described in the proximal colon, the molecular identity of the K+ channel(s) that mediates aldosterone-induced K+ secretion in the proximal colon is not known, nor is the involvement of NKCC1 in the process. Experiments were therefore designed to test the hypothesis that aldosterone up-regulates NKCC1 in the proximal colon to support BK channel-mediated K+ secretion, congruent with its effects in the distal colon. Using dietary Na+ -depletion as a model of secondary hyperaldosteronism in rats, we found that NKCC1 protein expression in the proximal colonic mucosa was enhanced 2-fold (p < 0.05) by Na+ depletion (i.e., aldosterone), whereas mRNA abundance was unaffected (p = 0.32). Surprisingly, electrogenic K+ secretion was not detectable by short-circuit current (ISC ) measurements, in response to either basolateral bumetanide (NKCC1 inhibitor) or luminal Ba2+ (non-selective K+ channel blocker), despite enhanced K+ secretion in Na+ -depleted vs. normal rats, as measured by 86 Rb+ fluxes under voltage-clamped conditions (net K+ secretion = -2.1 ± 0.6 vs -0.4 ± 0.2 mEq/cm2 .hr; p < 0.05). Expression of both BK and intermediate conductance K+ (IK) channels was also found to be unaltered by dietary Na+ depletion, at both the protein and mRNA level. However, basal ISC (146.7 ± 16.8 vs. 93.7 ± 8.7 mA/cm2 ; p < 0.05), as well as bumetanide-sensitive, Ca2+ - and cAMP-stimulated ISC (i.e., Cl- secretion) were significantly enhanced by dietary Na+ depletion (ΔISC = 239.6 ± 5.6 vs. 105.7 ± 33.5 and 154.3 ± 8.7 vs. 115.3 ± 8.7 mA/cm2 for Ca2+ and cAMP, respectively; p < 0.05 for both). In parallel to this was an increase in cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel protein expression (~1.7-fold; p < 0.05). We therefore reject our original hypothesis and conclude that NKCC1-dependent secretory pathways are differentially regulated by aldosterone in proximal and distal colon, where they are biased toward Cl- and K+ secretion, respectively. Development of therapeutic strategies in treating pathologies related to aberrant or altered colonic K+ /Cl- transport - such as pseudo-obstruction, ulcerative colitis, or end-stage renal disease (ESRD) - may benefit from these findings.

PMID:35553347 | DOI:10.1096/fasebj.2022.36.S1.R4485

FASEB J. 2022 May;36 Suppl 1. doi: 10.1096/fasebj.2022.36.S1.R6165.

ABSTRACT

One-third of patients with severe lung infection by influenza A virus (IAV) develop secondary lung infection by inhaled S. aureus(SA), leading to mortality in 30% of cases. It is unclear how IAV promotes secondary SA infection in lung alveoli, where fluid barrier dysfunction causes fatal acute lung injury (ALI). Our objective is to define alveolar responses to IAV that promote SA infection, leading to SA-induced alveolar damage and mortality. Our hypothesis is IAV lung infection blocks alveolar wall liquid (AWL) secretion, a homeostatic alveolar function that defends against alveolar stabilization of inhaled particles. AWL secretion depends on function of the alveolar epithelial cystic fibrosis transmembrane conductance regulator (CFTR) ion channel. Our methods include optical imaging of live, intact mouse lungs and mouse survival determinations. Mice were untreated or intranasally instilled with IAV (2000 PFU, A/PR/8/34). We excised, inflated, and blood-perfused the lungs at 24h, then viewed live alveoli by confocal microscopy. Alveolar airspaces were microinstilled separately with: (A) the fluorescent cytosolic dye, calcein-AM; (B) the fluorescent AWL tracer, tetramethylrhodamine-conjugated dextran (70 kD); (C) the CFTR potentiator, ivacaftor; and (D) the SA toxin, alpha hemolysin (Hla). For survival studies, IAV-infected mice were intranasally instilled with SA (USA300, 1x10^8 CFU) at 24h, then observed for 72h. Mice were given intraperitoneal injections of ivacaftor or vehicle at 4, 24, and 48h after SA instillation. Analysis was by t test (n=3) and log rank. Our results follow. Calcein fluorescence indicated the alveolar epithelium of IAV-infected lungs was viable. In alveoli of untreated lungs, airspace dextran fluorescence decreased over 1h, indicating dextran was progressively diluted by AWL secretion. However, in IAV-infected lungs, dextran fluorescence was steady (P<0.05), indicating IAV blocked AWL secretion. Since alveolar ivacaftor pretreatment restored dextran loss (P=NS versus untreated), we interpret IAV blocked AWL secretion in a CFTR-dependent manner, and CFTR potentiation rescued AWL secretion. Hla microinstillation into alveoli of IAV-infected lungs caused loss of epithelial calcein fluorescence, but alveolar pretreatment with ivacaftor blocked the loss (P<0.05). We interpret that rescue of AWL secretion protected against Hla-induced alveolar damage, probably by promoting Hla clearance and disrupting Hla-epithelial interactions. IAV-infected mice treated with ivacaftor were protected from SA-induced mortality (60% and 0% in vehicle- versus ivacaftor-treated mice, respectively; P<0.05; N=10 each). We conclude that IAV disrupted AWL secretion, causing retention of airspace contents and augmenting Hla-induced alveolar damage. Rescue of AWL secretion blocked Hla-induced alveolar damage and increased survival in IAV-SA coinfected mice. We propose strategies that rescue AWL secretion may represent a new therapeutic approach for ALI caused by IAV-SA coinfection.

PMID:35553259 | DOI:10.1096/fasebj.2022.36.S1.R6165

FASEB J. 2022 May;36 Suppl 1. doi: 10.1096/fasebj.2022.36.S1.R2204.

ABSTRACT

Cystic fibrosis (CF) is a genetic disease caused by absence of the cystic fibrosis transmembrane conductance regulator (CFTR) affecting multiple organs. CFTR is an anion transporter that moves chloride and bicarbonate ions to the apical side of the airway epithelial surface. CFTR deficiency results in decreased airway surface liquid depth and abnormally acidic airway pH, leading to decreased mucociliary transport and subsequent airway infections with pathogens such as Pseudomonas aeruginosa. While a major anion transporter in the epithelium, CFTR is not the only channel responsibly for efflux of chloride to the apical surface of the airway. Additional chloride transporters such as TMEM16A, SLC26A9, and CLC2 are present on the airway and have been detected to be potential disease-modifying genes. Recently, these transporters have been postulated to be therapeutic targets for supportive therapies in CF and other airway diseases. Here, we sought to determine the role of these chloride transporters in the development of lung disease of the CFTR-/- rat model. Short-circuit current data suggest that the largest portion of chloride transport function in the rat lung is caused by CFTR; however there remains some residual non CFTR-dependent current that may be due to additional chloride transporters. To determine which were important in the airway, CFTR-/- rats at 1, 3, and 6 months of age were assayed for mRNA and protein concentrations of TMEM16A, SCL26A9, and CLC2 compared to their wild-type (WT) littermates. Tracheae from these rats were excised and mucus particles tracked to determine mucociliary transport. We also assayed rats before and after lung infection with P. aeruginosa. Initial results indicated that TMEM16A and SLC26A9 mRNA and protein expression are increased in CFTR-/- compared to their WT littermates at each age tested and remain elevated compared to control following infection with P. aeruginosa. CLC2 has increased expression in CFTR-/- rats only following infection with P. aeruginosa. We found no correlation between expression of these transporters and mucociliary transport rates. However, we believe that results obtained suggest that these transporters may be exploited via agonists to improve airway surface liquid depth and pH. Future studies will examine the role of these transporters in correction of CF airway pathophysiology.

PMID:35553257 | DOI:10.1096/fasebj.2022.36.S1.R2204

FASEB J. 2022 May;36 Suppl 1. doi: 10.1096/fasebj.2022.36.S1.R5228.

ABSTRACT

Goblet cell metaplasia and/or mucus hypersecretion are hallmark features of multiple lung diseases, including asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis and pulmonary fibrosis. Interleukin 1β (IL-1B) is a proinflammatory mediator that increases expression of the major gel forming mucins in the airways, Muc5b and Muc5ac, in part through activation of the cAMP response element binding protein (Creb) transcription factor. IL-1B also induces Spdef, a key regulator of goblet cell differentiation from airway club cells. Here we tested the hypothesis that elimination of murine club cell Creb mitigates muco-obstructive phenotypes induced by IL-1B. Mice with floxed Creb1 were bred to Scgb1atm1(cre/ERT)Blh to generate mice with conditional loss of Creb1 from club cells. Wild-type and transgenic male mice (7-9/group) littermates received intranasal IL-1B (0.5 ng/inhalation) or saline (control) for four consecutive days, with tamoxifen administration occurring on days 1 and 3 to induce Cre recombinase expression. Periodic acid-Schiff staining in lung samples showed that IL-1B increased the number of goblet cells in central airways compared to saline controls. This effect was blunted by conditional loss of Creb1. Similarly, flexiVent measurements demonstrated that IL-1B increased airway resistance (R), Newtonian resistance (Rn), tissue elastance (ERS), tissue damping (G) and tissue elasticity (H). Loss of club cell Creb1 mitigated all effects of IL-1B on airway mechanics. Additionally, though the percentage of granulocytes in the bronchoalveolar lung lavage fluid was not increased by IL-1B, loss of club cell Creb1 decreased the percentage of granulocytes in the bronchoalveolar lung lavage fluid independent of treatment (i.e., saline or IL-1B). These findings suggest that the muco-obstructive effects of IL-1B are dependent upon club cell Creb1 and provide novel insight for therapeutic strategies.

PMID:35553253 | DOI:10.1096/fasebj.2022.36.S1.R5228

FASEB J. 2022 May;36 Suppl 1. doi: 10.1096/fasebj.2022.36.S1.R3636.

ABSTRACT

Cystic fibrosis transmembrane conductance regulator (CFTR) plays crucial role in renal cyst expansion via increase in fluid accumulation. Evidence suggests that inhibition of CFTR retard cyst development and enlargement in polycystic kidney disease (PKD). Here we investigated the pharmacological effect of pinostrobin, a bioactive natural flavonoid on CFTR-mediated Cl- secretion and renal cyst expansion in in vitro model; human autosomal dominant polycystic kidney disease (ADPKD) cell line and Type I Mardin Darby Canine Kidney (MDCK) cells. Treatment with pinostrobin (10 and 50 µM) significantly reduces MDCK cyst formation and enlargement in concentration-dependent manner. Pinostrobin reduces the number of renal cyst colonies corresponding with a decrease in cell proliferation and extracellular signal-regulated kinases (ERK) signaling. Additionally, pinostrobin retarded cyst expansion via inhibition on CFTR-mediated chloride secretion. The inhibitory effect of pinostrobin was not due to the decrease in cell viability and activity of Na+ -K+ -ATPase. Our findings define that pinostrobin might be the candidate for further study as the therapeutic agents for PKD treatment.

PMID:35553007 | DOI:10.1096/fasebj.2022.36.S1.R3636

FASEB J. 2022 May;36 Suppl 1. doi: 10.1096/fasebj.2022.36.S1.R4308.

ABSTRACT

The COVID-19 pandemic brought to light the continued issues in access to research opportunities. Many of our undergraduate students at research-intensive institutes lost their ability to go into labs to gain experience. However, students at most smaller colleges and universities faced these challenges to bring research opportunities to students long before the pandemic. Thus, our lower-cost community colleges and higher education institutes with diverse students lack equity when it comes to research investments. This highlights the need for bringing research into diverse institutes using novel approaches. Over the past ten years, our team has built a strategy for integrating genomic and molecular bioinformatics tools into undergraduate opportunities. Developing bioinformatics training material, professor training opportunities, summer research opportunities, and organized large scale research projects, our team has developed a distributed research network. Our goal is to bring genomic and bioinformatic literacy to early undergraduate training to increase STEM retention while improving research equity issues. Having students work on characterizing challenging clinical variants, known as Variants of Uncertain Significance (VUS), brings novel research projects to students while filling the growing needs of clinical variant characterizations. At the core training, we focus on bringing tools to professors and instructors, lowering the initiation efforts to performing bioinformatics research. These include the optimization of protein homology modeling, molecular dynamic simulations through analysis with a few mouse clicks (or two lines of code for Linux users), 3D protein printing, deep evolutionary profiling with hundreds of species using user-friendly tools, assessing expression from the NCBI SRA, and integrating genomic databases (GTEx, Human Protein Atlas, Geno2MP, gnomAD, Comparative Toxicogenomics Database, PharmGKB, Open Targets Genetics). Once in the hands of professors, they have implemented these tools into independent research projects for their students, coursework design such as bioinformatics classes, and research clubs. Over the past ten years, we have thus impacted hundreds of students with these tools. This can best be highlighted by the student's successes integrating the tools into publications on the NMDA receptors (PMID:34726335), CFTR database for cystic fibrosis (PMID: 32734384), NAA10 variant analysis (PMID: 33335012), multiple sclerosis genetics (PMID: 31482761), COVID-19 immune response (PMID:34335605), the SARS-CoV-2 evolution/structural dynamicome (PMID: 32587094), CCR5 role in diverse phenotypes and bioethics (accepted), and SOX gene developmental biology (in review). Thus, the development of tools and strategies and the distribution of research projects can reach students and faculty at any institute, bringing equitable research opportunities to those who traditionally do not have many opportunities.

PMID:35552883 | DOI:10.1096/fasebj.2022.36.S1.R4308

FASEB J. 2022 May;36 Suppl 1. doi: 10.1096/fasebj.2022.36.S1.R5499.

ABSTRACT

Burkholderia cenocepaciais an opportunistic pathogen posing risk among individuals with cystic fibrosis or who are immune compromised. The crystal structure of a putative hydroxybutyrate dehydrogenase (HBDH), based on sequence analysis, from Burkholderia cenocepaciahas been solved by the Seattle Structural Genomics Center for Infectious Disease and deposited in the Protein Database (PDB ID 4TRR). We sought to determine if the protein is an HBDH and to determine the kinetic parameters of the enzyme. HBDHs catalyze the reversible conversion of acetoacetate to hydroxybutyrate utilizing the cofactor NAD(H). The SSGCID kindly provided us the construct encoding a His-tagged BcHBDH. The protein was overexpressed in E. coli, purified to approximate homogeneity using nickel affinity chromatography, and activity assayed spectrophotometrically. The putative BcHBDH is confirmed to have HBDH activity and the kinetic parameters for the enzyme were determined in both the direction of acetoacetate and hydroxybutyrate formation. BcHBDH has a catalytic efficiency of 2.85x104 mM-1 sec-1 and 1.21x105 mM-1 sec-1 in the direction of hydroxybutyrate formation for acetoacetate and NADH, respectively. In the direction of acetoacetate formation, the enzyme has a catalytic efficiency of 4.1x103 mM-1 sec-1 and 2.86x104 mM-1 sec-1 for hydroxybutyrate and NAD+ , respectively. By comparing both the structure and kinetic parameters to known kinetically characterized and crystallized HBDHs, we conclude the protein from Burkholderia cenocepacia is an HBDH.

PMID:35552584 | DOI:10.1096/fasebj.2022.36.S1.R5499

FASEB J. 2022 May;36 Suppl 1. doi: 10.1096/fasebj.2022.36.S1.R3248.

ABSTRACT

Variations in salinity are among the main physical parameters that drive the capacity of fish to survive and thrive in a range of environments. Acclimation to changes in environmental salinity is regulated by the interplay of local osmotically-induced cellular responses and the systemic regulation by the neuroendocrine system, which together, direct ion extrusion or uptake responses via the gill and other osmoregulatory tissues. Many euryhaline teleost fish are native to waters in which salinity varies tidally between that of fresh water (FW) and seawater (SW), such as estuaries. The physiology of salinity acclimation of euryhaline teleosts has been studied extensively in steady-state salinities, and after one-way transfers between steady-state salinities. Fewer studies, however, have addressed salinity regimes that reflect or simulate the continuous dynamic changes by which euryhaline fishes may be subject to in a native environment. Experimentally, the simulation of a changing environment was obtained by rearing fish in salinities that changed between those of FW and SW every six hours, in a tidal regime (TR). An overview of the main differences in the regulation of transcripts involved in ion balance between euryhaline Mozambique tilapia, Oreochromis mossambicus, responding to TR and those acclimating to steady-state salinities is provided. Transcripts analyzed include branchial Na+/K+-ATPase (nka), Na+, Cl- co-transporter (ncc), Na+/H+ antiporter 3 (nhe3), 2Cl- co-transporter (nkcc), cystic fibrosis transmembrane conductance regulator (cftr), and aquaporin 3 (aqp3). In particular, both cftrand aqp3were highly sensitive to changes in salinity in fish acclimated to a TR, indicating indispensable roles in rapidly maintaining hydromineral balance. The pituitary hormones, prolactin and growth hormone, known to play osmoregulatory roles in Mozambique tilapia, were also compared along with their receptors between TR and steady-state salinity paradigms. Specifically, through the dynamic changes in hormone receptor transcription observed in the TR rearing paradigm, the endocrine control of osmoregulatory outcomes appears shifted from systemic to local regulation at the level of target tissues. Together, these studies indicate that fish are able to compensate for broad and frequent changes in external salinity while keeping osmoregulatory parameters within a narrow range.

PMID:35552045 | DOI:10.1096/fasebj.2022.36.S1.R3248

FASEB J. 2022 May;36 Suppl 1. doi: 10.1096/fasebj.2022.36.S1.R5972.

ABSTRACT

SLC26A9 (solute carrier family 26, member 9) is highly expressed epithelial cells of the respiratory tract, stomach, pancreas and kidney, and acts as a Cl- /HCO3 - exchanger. SLC26A9 plays a pivotal role in HCO3 - secretion in lung, and emerging evidence suggests that SLC26A9 is a therapeutic target for cystic fibrosis (CF) and potentially other mucosal obstructive pulmonary diseases. In this study, a cell-based high-throughput screening (HTS) assay was established and performed to identify highly potent and selective small molecule inhibitors of SLC26A9. Screening of 30,000 synthetic small molecules identified three novel SLC26A9 inhibitors. Unlike the previous non-selective SLC26A9 inhibitors, the novel SLC26A9 inhibitors potently and selectively inhibited anion exchange activity of SLC26A9. The most potent inhibitor, A9-301, significantly blocked Cl- /I- exchange activity of SLC26A9 (IC50 of ~100 nM) without affecting other anion exchanges and chloride channels, including SLC26A3 (DRA), SLC26A4 (pendrin), SLC26A7, CFTR and ANO1. These results suggest that the novel potent and selective inhibitors of SLC26A9 are useful tools for pharmacological dissection of SLC26A9 and may be potential therapeutic candidates for airway inflammatory diseases.

PMID:35551984 | DOI:10.1096/fasebj.2022.36.S1.R5972

FASEB J. 2022 May;36 Suppl 1. doi: 10.1096/fasebj.2022.36.S1.R5249.

ABSTRACT

The cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel expressed in epithelia and other tissues, is compromised by genetic mutations that cause cystic fibrosis (CF). Although CFTR is reportedly permeant to both Cl¯ and HCO3¯, high throughput screening studies identified drug scaffolds that enhanced Cl¯ conductance of mutated CFTR by 'correcting' processing deficiets and/or 'potentiating' aggregate conductance of channels in epithelial cell membranes while being agnostic to HCO3-. Ongoing work seeks to determine whether mutated forms of CFTR exhibit typical HCO3¯ conductance and to access clinically-used drugs for their effects on HCO3¯ conductance by wild-type and mutated CFTR. Fisher rat thyroid (FRT) cells, which form electrically tight epithelial monolayers, express no functionally detectable CFTR and which traffick exogenously expressed CFTR to both the apical and basolateral membranes, were employed to assess transepithelial anion conductance in modified Ussing-style flux chambers. Initial experiments employing FRT cells expressing human wtCFTR validated that forskolin-stimulated conductance, which was inhibited by selective CFTR blockers, was proportional to extracellular Cl¯ concentration over the range of ~70-140 mEq/l. Further, a clear increment in forskolin-stimulated conductance was observed when HCO3¯ (25 mEq/l) was incorporated into the media. VX770 potentiated forskolin-stimulated conductance, but had no effect in the absence of forskolin. Foskolin-stimulated and VX770-potentiated conductance was compared in base medium (100 mEq/l Cl-) supplemented with 25 mEq/l HCO3- or Cl- and bicarbonate (B) conductance was quantified relative to Cl- (C) conductance (i.e., gB/gC). Following forskolin exposure the gB/gC ratio for wtCFTR was 0.62±0.20, a value that decreased to 0.11±0.10 following exposure to VX770, suggesting that VX770 may negatively impact gB. Forskolin- and VX770-stimulated conductance across cells expressing F508del CFTR was nearly undetectable unless cells were pretreated with CFTR correctors. Following corrector exposure, forskolin stimulated and VX770 potentiated gC, but gB was virtually undetectable, suggesting that this variant may lack gB. Alternatively, forskolin stimulated little conductance across cells expressing G551D CFTR, but a substantial potentiation with VX770 was observed. Notably, the gB/gC ratio approached 1.0 for both foskolin stimulation and VX770 potentiation. Taken together, these results suggest that CFTR HCO3- conductance differs by disease-associated variant and that gB of some variants are sensitive to VX770. Ongoing experiments are designed to determine the optimal forskolin concentration to maximize VX770 potentiation. Initial outcomes suggest that there is interaction between the CFTR variant and forskolin concentration dependence such that VX770-potentiated conductance is maximized at different forskolin concentrations. Taken together, results from this investigation suggest that additional or alternative interventions may be required to optimize bicarbonate secretion and pH regulation in CF patients expressing alternative CFTR variants.

PMID:35551952 | DOI:10.1096/fasebj.2022.36.S1.R5249

FASEB J. 2022 May;36 Suppl 1. doi: 10.1096/fasebj.2022.36.S1.R3551.

ABSTRACT

Pneumonia is the most common cause of the acute respiratory distress syndrome (ARDS), a potentially fatal lung disease characterized by hyperinflammation and endothelial barrier failure. Infectious and inflammatory stimuli can cause rapid downregulation of cystic fibrosis transmembrane conductance regulator (CFTR), and inhibition of CFTR has been proposed to increase lung microvascular endothelial permeability in vitro. Here, we identify loss of lung microvascular endothelial CFTR as important pathomechanism in lung barrier failure in pneumonia-induced ARDS, delineate the molecular signaling pathway underlying this effect and identify CFTR potentiation as novel therapeutic strategy in ARDS. CFTR was downregulated in endothelial cells following Streptococcus pneumoniae(S.pn.) infection in human and murine lung tissue. Isolated perfused lungs revealed that CFTR inhibition increased endothelial permeability in parallel with intracellular Cl- and Ca2+ concentrations ([Cl- ]i , [Ca2+ ]i ). Inhibition of the Cl- sensitive with-no-lysine kinase 1 (WNK1) replicated the effect of CFTR inhibition on endothelial permeability and endothelial [Ca2+ ]i while WNK1 activation attenuated it. Endothelial [Ca2+ ]i transients and permeability in response to inhibition of either CFTR or WNK1 were prevented by inhibition of the cation channel transient receptor potential vanilloid 4 (TRPV4). Mice deficient in Trpv4 (Trpv4-/- ) developed less lung edema and protein leak than their wild-type littermates following infection with S. pn. Conversely, lungs of heterozygous Wnk1-deficient mice (Wnk1+/- ) showed spontaneous leak. The CFTR potentiator ivacaftor prevented CFTR loss and reduced endothelial leak in response to pneumolysin (PLY), a key S.pn. virulence factor, or plasma from COVID-19 patients in vitro, and prevented lung edema and protein leak after S. pn.infection in vivo. Lung infection causes rapid loss of CFTR that promotes lung edema formation through intracellular Cl- accumulation, inhibition of WNK1 and subsequent disinhibition of TRPV4, resulting in endothelial Ca2+ influx and vascular barrier failure. Ivacaftor prevents CFTR loss and may thus present a promising therapeutic strategy in ARDS due to severe pneumonia including COVID-19.

PMID:35551909 | DOI:10.1096/fasebj.2022.36.S1.R3551