Inhibition of NFκB by the natural product Withaferin A in cellular models of Cystic Fibrosis inflammation
Cystic Fibrosis (CF) is one of the most common autosomal genetic disorders in humans. This disease is caused by mutations within a single gene, coding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The phenotypic hallmark of CF is chronic lung infection and associated inflammation from opportunistic microbes such as Pseudomonas aeruginosa (PA), Haemophilus influenzae, and Staphylococcus aureus. This eventually leads to deterioration of lung function and death in most CF patients. Unfortunately, there is no approved therapy for correcting the genetic defect causal to the disease. Hence, controlling inflammation and infection in CF patients are critical to disease management. Accordingly, anti-inflammatory agents and antibiotics are used to manage chronic inflammation and infection in CF patients. However, most of the anti-inflammatory agents in CF have severe limitations due to adverse side effects, and resistance to antibiotics is becoming an even more prominent problem. Thus, new agents that can be used to control chronic inflammation in CF are needed in the absence of a cure for the disease. Activation of the transcription factor NFκB through Toll-like receptors (TLR) following bacterial infection is principally involved in regulating lung inflammation in CF. NFκB regulates the transcription of several genes that are involved in inflammation, anti-apoptosis and anti-microbial activity, and hyper-activation of this transcription factor leads to a potent inflammatory response. Thus, NFκB is a potential anti-inflammatory drug target in CF. Screening of several compounds from natural sources in an in vitro model of CF-related inflammation wherein NFκB is activated by filtrates of a clinically isolated strain of PA (PAF) led us to Withaferin A (WFA), a steroidal lactone from the plant Withania Somnifera L. Dunal. Our data demonstrate that WFA blocks PAF-induced activation of NFκB as determined using reporter assays, IL-8 measurements and high-content fluorescent imaging of NFκB subunit p65 translocation. Since the airways of CF patients can be specifically targeted for delivery of therapeutics, we propose that WFA should be further studied as an anti-inflammatory agent in models of CF related inflammation mediated by NFκB.
KeywordsCystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator Cystic Fibrosis Patient Cystic Fibrosis Transmembrane Conductance Regulator Mutation Luciferase Reporter Activity
Cystic Fibrosis (CF) is one of the most common lethal autosomal recessive diseases in humans. It is caused by mutations within a single gene, coding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein (reviewed in ). Loss of lung function causes over 90% of all CF deaths [2, 3], which is brought about by chronic bacterial infections involving drug-resistant pathogenic strains of Pseudomonas aeruginosa (PA), Haemophilus influenzae and Staphylococcus aureus [3, 4, 5] among others. Chronic and uncontrolled stimulation of cellular signaling by bacterial products through toll-like receptors (TLRs) lead to hyper-activation of the transcription factor NFκB and over-expression of a number of pro-inflammatory cytokines [6, 7, 8]. Consequently, an overwhelming number of neutrophils and macrophages are attracted to the site of infection and these cells release proteases and other agents that cause structural damage to the airways. Anti-inflammatory agents are used to manage lung inflammation in CF, but have adverse effects  that limit their use. Thus, there is a need to identify drugs with limited toxicity to treat lung inflammation in CF .
Screening of natural products with purported anti-inflammatory activity led us to Withaferin A (WFA), a steroidal lactone isolated from the herb Withania somnifera (also known as Indian Ginseng and Ashwagandha), which is widely used in traditional Indian medicine as an anti-inflammatory agent . Recent reports indicate that this natural product is an inhibitor of NFκB activity [12, 13]. The overall goal of this study was to characterize the effect of this compound on NFκB in cellular models of CF-related inflammation. In our studies, filtrates of PA isolated from a CF patient were used. This is an established method to experimentally induce inflammation in the field of CF research and is relevant to airway inflammation noted in patients [14, 15]. Inflammation in CF is caused by a complex mixture of bacterial products including secreted toxins, lipoproteins, lipopolysaccharides and bacterial DNA . The filtrates used in our studies isolated from post log-phase cultures of PA contain many of these harmful agents. These products differentially activate various TLRs expressed in airway epithelial cells and ultimately increase expression of pro-inflammatory genes regulated by NFκB .
Unless specified, all reagents were purchased from Sigma Aldrich (St. Louis Missouri). The KKLEB immortalized CF airway cell line homozygous for ΔF508 mutation and the CF 14 clinically isolated mucoid strain of PA (originally from the laboratory of Dr. M.C. Wolfgang, University of North Carolina) were donated by Dr. S. Randell (University of North Carolina) . HEK 293 cells were obtained from ATCC (Manassas, VA). Cells were maintained in DMEM/F12 medium with 10% fetal bovine serum and antibiotics. A NFκB reporter plasmid was constructed as follows: Complementary oligonucleotides bearing NFκB consensus DNA-binding sequence (5'-gctagc tgg gga ctt tcc gct ggg gac ttt ccg ctg ggg act ttc cgc tgg gga ctt tcc gct ggg gac ttt ccg c aagctt-3') were synthesized with flanking NheI and HindIII sites (underlined), annealed and introduced into the pGL4.26 (luc2/minP/Hygro) vector (Promega, Madison, WI). The construct was linearized with Bsu36I and transfected into HEK293 cells using Fugene HD (Roche Diagnostics, Indianapolis, IN). Clonal cell-lines stably expressing the construct were identified following selection in Hygromycin-containing media and tested for NFκB-mediated induction of luciferase reporter activity using recombinant TNF-α and filtrates of a clinically isolated mucoid strain of PA (PAF) from a CF patient (data not shown). A stable cell line designated HEK293/NFκB-luc was used for the reported experiments. For transient transfection assays, KKLEB cells were batch transfected using Fugene HD reagent in suspension and subsequently plated out into 24-well plates. This approach nullified the need to use a second reporter gene for data normalization. Unless otherwise noted, cells were allowed to incubate overnight and then induced with PAF for 24 hr in serum-containing media. Typically, cells were pre-incubated with WFA (Chromadex, Santa Ana, CA) for 2 hr and then stimulated with PAF that were prepared essentially as described previously . Briefly, the clinically isolated mucoid strain of PA was grown for 72 hr in LB media. The supernatant from the culture was removed by centrifugation, boiled for 10 min to inactivate proteolytic activity, aliquoted, and stored at -80°C. Luciferase assays were conducted using a kit obtained from Promega in a TECAN plate-reader. Quantification of IL-8 in media was performed using a commercially available sandwich ELISA kit (Biolegend Inc., San Diego, CA). For the NFκB subunit p65 translocation studies using immunofluorescence microscopy, KKLEB cells were seeded in black optical-bottom 96-well plates and treated as described above with PAF and WFA. Following treatment, cells were fixed with 3.7% formaldehyde in PBS, and fluorescently labeled using a commercially available kit (NFκB activation HCS kit, Thermo Scientific, Waltham, MA). Fluorescent images were acquired at 20× magnification using a Discovery 1 automated fluorescent microscope (MDS Analytical Devices) with filter sets appropriate for FITC (for p65 detection) and DAPI (nuclear stain). Six images were analyzed per group resulting in analysis of roughly 300 cells per treatment. Nuclear translocation of p65 was measured using the enhanced translocation module from the Metaxpress image analysis software provided with the instrument. Input settings delineating cell "compartment" (nucleus) and "regions for measurement" (cytoplasm) were entered as follows: Compartment-width = 10 μm, intensity above background = 200 gray levels, minimum area = 5 μm2, and maximum area = 1000 μm2. Regions for Measurement (RFM) were entered as follows: Inner region distance from edge = 1 μm, outer region distance from edge = 1 μm, outer region width = 6 μm, background correction = none. Cells were scored as positive for nuclear translocation of p65 if the correlation coefficient was 0.75 or greater. Cell viability was monitored using the Cell-Titer Glo Luminescent Cell Viability Assay (Promega Corporation) following the manufacturer's suggestion. All concentrations used for our studies were non-cytotoxic to the cells (data not shown) under the experimental conditions.
CF as a disease is largely limited to Caucasians. According to a recent report, ~30% of patients suffering from progressive medical conditions including CF use complementary and alternative approaches to supplement conventional therapies . We are intrigued by this finding, as there are many promising anti-inflammatory and anti-bacterial ethnopharmacological agents that have not been adequately studied in the context of diseases that are atypical in native populations, such as CF, where they may provide a benefit. Thus, our long-term goal is to develop strategies and platforms to test such agents in CF and other diseases. Our studies with WFA demonstrate the potential of natural products in preventing inflammation in CF mediated by NFκB. WFA itself is toxic to cells at high concentrations  but not at the concentrations used in this study. However, other less toxic withanolides  from W. somnifera may be useful as early leads to treat inflammation in CF. Alternatively, structure activity studies of WFA using medicinal chemistry may lead to compounds that inhibit NFκB activity without undesirable side effects. Further, targeted delivery of anti-NFκB agents to the airways of CF patients is possible using inhaled aerosols , which would restrict their effects largely to target tissues where this transcription factor is reported to be hyper-activated. We propose to explore some of these possibilities in future studies.
This work was supported by a grant (1R21NSO61743-01) to RM from NIH. Additional financial support for the project was provided by the Research Triangle Institute.
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