Aflatoxins are mycotoxins secreted by Aspergillus flavus, which can colonize the respiratory tract and cause fungal rhinosinusitis or bronchopulmonary aspergillosis. A. flavus is the second leading cause of invasive aspergillosis worldwide. Because many respiratory pathogens secrete toxins to impair mucociliary immunity, we examined the effects of acute exposure to aflatoxins on airway cell physiology. Using air-liquid interface cultures of primary human sinonasal and bronchial cells, we imaged ciliary beat frequency (CBF), intracellular calcium, and nitric oxide (NO). Exposure to aflatoxins (0.1 to 10 μM; 5 to 10 minutes) reduced baseline (~6–12%) and agonist-stimulated CBF. Conditioned media (CM) from A. fumigatus, A. niger, and A. flavus cultures also reduced CBF by ~10% after 60 min exposure, but effects were blocked by an anti-aflatoxin antibody only with A. flavus CM. CBF reduction required protein kinase C but was not associated with changes in calcium or NO. However, AFB2 reduced NO production by ~50% during stimulation of the ciliary-localized T2R38 receptor. Using a fluorescent reporter construct expressed in A549 cells, we directly observed activation of PKC activity by AFB2. Aflatoxins secreted by respiratory A. flavus may impair motile and chemosensory functions of airway cilia, contributing to pathogenesis of fungal airway diseases.
Mucociliary clearance (MCC) is the primary physical defense of the respiratory tract against inhaled pathogens1. When MCC fails, respiratory infections significantly impair patient quality of life1. Upper respiratory infections (URIs) often result in chronic rhinosinusitis (CRS), a complex syndrome involving stasis of sinonasal secretions and inflammation. CRS causes ~8 billion dollars of yearly direct healthcare costs in the US alone2 and accounts for ~20% of adult antibiotic prescriptions3,4,5, making CRS a major driver of antibiotic resistance6,7. URIs also “seed” lower respiratory infections and/or exacerbate lung diseases such as cystic fibrosis or chronic obstructive pulmonary disease8. It is important to better understand the complex pathogenesis of respiratory infections to generate novel therapies and improve outcomes for these airway diseases.
Fungi of the Aspergillus genus are ubiquitously found in nature9,10. While ~90% of Aspergillus are non-pathogenic, some are opportunistic pathogens that colonize the human respiratory tract9,10, including A. fumigatus, A. niger, and A. flavus. Sensitization to colonizing Aspergillus can cause allergic bronchopulmonary aspergillosis11 or allergic fungal rhinosinusitis12. Moreover, in immunocompromised individuals (e.g., chemotherapy patients) or those with damaged airways (e.g., CF or CRS patients) Aspergillus infections can invade the mucosa9. Acute or chronic invasive fungal rhinosinusitis or pulmonary aspergillosis requires aggressive antifungal and/or surgical intervention to prevent mortality. It is particularly critical to understand the interactions of Aspergillus with the airway epithelium to understand the pathogenesis of fungal respiratory infections.
Many respiratory pathogens secrete factors that impair MCC, including Pseudomonas aeruginosa13,14,15 and Streptococcus pneumoniae16. The fungus A. fumigatus also secretes gliotoxin, fumagillin, and helvoilic acid10,17,18,19, which can slow ciliary beat frequency (CBF)18. The mechanism(s) of their ciliotoxicities are not yet well defined, but conditioned media (CM) from clinical isolates of A. fumigatus as well as sputum from patients with pulmonary aspergillosis impair cilia function and damage epithelial tissue17,18. While A. fumigatus is the most common pathogenic Aspergillus, A. flavus is the second-leading cause of invasive aspergillosis20,21. A. flavus infection is rare in the US and Europe. However, bronchiopulmonary and sinonasal apergillosis from A. flavus is common in India, Africa, South East Asia, and the Middle East, possibly due to an increased ability of A. flavus to thrive in arid conditions20. A. flavus in the upper respiratory tract is often associated with chronic granulomatous sinusitis. A. flavus is of importance because it produces aflatoxins, which are among the most potent naturally-occuring hepatic carcinogens known19. Ingestion of contaminated foods results in metabolism (“activation”) of aflatoxins in the liver into reactive DNA-damaging epoxides that cause hepatic necrosis, cirrhosis, and/or carcinoma22,23.
Inhalation of aflatoxins has been associated with occupations involving exposure to environmental molds24, such as grain processing. However, the effects of inhaled aflatoxins or aflatoxin-producing fungi on the airway epithelium are not well characterized. There is some evidence that airway cells can activate aflatoxins in vitro4,25 and in vivo26,27,28, though the link between aflatoxin exposure and human lung cancer is unclear. However, aflatoxins can increase protein kinase C (PKC) activity in some cell lines in vitro29,30,31. Because PKC can decrease CBF32,33 through phosphorylation of ciliary proteins32,33, we hypothesized that aflatoxins may have acute effects on MCC that contribute to A. flavus pathogenesis.
Aflatoxin B2 Decreases CBF in a PKC-Dependent Manner
We examined epithelial responses to a common aflatoxin, aflatoxin B2 (AFB2). AFB2 (Fig. 1a) was chosen as the model aflatoxin for testing in this study because it has less carcinogenicity than AFB134,35 and thus should have less DNA-damaging nonspecific toxic effects. We utilized air-liquid interface cultures (ALIs) derived from human sinonasal and bronchial epithelial cells36. ALIs mimic the polarized respiratory epithelium with well differentiated ciliated and goblet cells37. High-speed imaging was used to track changes in CBF. Acute mucosal exposure (apical side only) of sinonasal ALIs to AFB2 (1 μM and 10 μM) significantly decreased basal CBF after only 5 minutes, while vehicle (DMSO) had no effect (Fig. 1b,c). The protein kinase C (PKC) inhibitor Gö698338 (10 μM; 5 min apical pre-treatment before experiment) significantly blunted the AFB2-mediated inhibition (Fig. 1d). AFB1 had nearly identical effects (Fig. 1e). Results are summarized in Fig. 1f,g. No additive effects of AFB2 were observed when sinonasal ALIs were pre-treated with the phorbol ester phorbol-12-myristate-13-acetate (PMA), nor was CBF further reduced when PMA was added to ALIs pre-treated with AFB2 (Fig. 1h), supporting the hypothesis that AFB2 reduces CBF through a PKC-dependent pathway.
We noted that short-term exposures to AFB2 and AFB1 also impaired activation of CBF in response to the purinergic agonist ATP (Fig. 1b,d), an important signaling molecule in the airway33. We thus carried out a detailed examination of the effects of AFB2 on stimulated CBF using several physiologically important agonists after 10 min exposure to 0.5 μM AFB2. AFB2 inhibited CBF during stimulation with 1 μM ATP (added apically; Fig. 2a), 10 μM isoproterenol (added apically; Fig. 2b), and 10 μM VIP (added basolaterally; Fig. 2c). CBF reductions (summarized in Fig. 2d) were blocked by the PKC inhibitors Gö6983 and calphostin C38. AFB2 exposure also reduced CBF increases in response to a mechanically-simulated “sneeze” (Fig. 2e), which stimulates CBF through apical ATP release and downstream calcium signaling36.
When we examined ALIs grown from human bronchial epithelial (HBE) cells, we found that AFB2 similarly reduced both basal and ATP-stimulated CBF via a PKC-dependent mechanism (Fig. 3). Interestingly, when we examined ALI cultures derived from mouse nasal septum, we found that AFB2 inhibited basal CBF but not ATP-stimulated CBF (Supplementary Fig. S1), reflecting a species-specific difference.
AFB2 Acts Independently of Calcium
Calcium is a master regulator in airway cells, controlling both ion transport39 as well as CBF32. Data above show that AFB2 reduces CBF in human ALIs in response to both ATP and the sneeze puff, which both require intracellular calcium, as well as VIP, which acts independently of calcium through cyclic AMP (cAMP) in these cells40. Thus, we hypothesized that AFB2 likely has direct effects on cilia function, possibly through PKC phosphorylation of cilia proteins, as previously described33, rather than by indirectly affecting calcium levels. However, because many isoforms of PKC are regulated by calcium33, we examined if AFB2 affects baseline or stimulated calcium signaling. We examined changes in intracellular calcium concentration in sinonasal ALIs loaded with the calcium-sensitive indicator fluo-4 during exposure to 10 μM AFB2. AFB2 had no detectible effect on intracellular calcium, nor did it affect the magnitude or kinetics of ATP-induced calcium signaling (Supplementary Fig. S2), supporting the hypothesis that AFB2 activates PKC independently of calcium.
AFB2 Acts Independently of the Y2 Neuropeptide Y Receptor
Neuropeptide Y (NPY) is one of the few neurotransmitters known to reduce CBF through Y2 receptor activation of PKC in primary human tracheal and bronchial ciliated cells41. In sinonasal ALIs, NPY decreased basal CBF by ~10% through a mechanism blocked by both the Y2 antagonist BIIE-0246 and Gö6983 (Fig. 4a,c). CBF was also reduced by the Y2 agonist NPY-(16–36) but not the Y1 agonist [Leu31,Pro34]-NPY (Fig. 4b,c). No additive effects were observed when NPY was added after AFB2 (Fig. 4d), suggesting they partially share the same pathway. However AFB2 reduction of CBF was not blocked by BIIE-0246, the broad spectrum neuropeptide receptor inhibitor antagonist G42, or the phospholipase C inhibitor U73122 (Fig. 4e). These data demonstrate that AFB2 functions independently of the Y2 receptor and likely other neurotransmitter receptors.
Exposure to A. flavus conditioned media (CM) reduces CBF via a PKC- and aflatoxin-dependent pathway
We tested whether conditioned medium (CM) from a known aflatoxin-producing strain of A. flavus could similarly reduce respiratory CBF. Experiments were carried out in sinonasal ALIs at 12.5% and 25% (Fig. 5a,b) and confirmed in bronchial ALIs at 25% (Fig. 5c,d). A. flavus CM significantly reduced baseline CBF after 60–75 min and significantly blunted ATP-induced CBF increase (Fig. 5b,d). While the kinetics of the CM-induced reduction in CBF was slower than observed with purified aflatoxin, these effects were nonetheless blocked by Gö6983 as well as when the A. flavus CM was pre-treated with anti-aflatoxin antibodies (recognizing both B and G group aflatoxins). These data strongly suggest that cultured A. flavus secretes aflatoxins at low concentrations that are nonetheless high enough to reduce airway CBF. We observed that CM from A. fumigatus and A. niger, which cannot secrete aflatoxins, was still observed to reduce CBF (Supplementary Fig. S3). The effects of A. fumigatus and A. niger CM were blocked by Gö6983 but not by anti-aflatoxin antibodies (Supplementary Fig. S3), suggesting that these species secrete other mycotoxins that can target PKC, perhaps including gliotoxin, fumagillin, and/or helvoilic acid. The identities and mechanisms of action of A. fumigatus and A. niger ciliotoxins remain to be determined in future studies.
AFB2 impairs sinonasal epithelial nitric oxide (NO) innate immune responses
Nitric oxide (NO) is an important mediator of host airway defense because it directly kills pathogens as well as increases CBF43,44. We recently showed that a bitter taste receptor, T2R38, is expressed in sinonasal epithelial cilia and drives NO production in response to bacterial acyl-homoserine lactone (AHL) quorum sensing molecules43,44,45. Because PKC can phosphorylate nitric oxide synthase (NOS) and prevent its activation46,47, we tested the effects of AFB2 on sinonasal NO production in response to the T2R38 agonist and Pseudomonas quorum sensing molecule N-3-oxo-dodecanoyl-L-homoserine lactone (C12HSL)43. Reactive nitrogen species (RNS) production was measured using the fluorescent indicator DAF-FM. RNS production was reduced by approximately one half in the presence of AFB2, and this effect was blocked by Gö6983 (Fig. 6a,b). To test if AB2-induced PKC activity had a general effect on NOS function or a specific effect on T2R38 function, we measured RNS production during global calcium elevation in cells exposed to the calcium ionophore ionomycin (10 μM) and the sarco/endoplasmic reticulum calcium ATPase (SERCA) inhibitor thapsigargin (10 μM). AFB2 also significantly reduced NO production under these conditions through a Gö6983-sensitive pathway (Fig. 6c,d), suggesting AFB2 has a direct effect on NOS activation rather than on T2R38 function.
AFB2 activates PKC in A549 cells in vitro
To further test the hypothesis that aflatoxins can activate PKC activity, we utilized a Förster resonance energy transfer (FRET)-based PKC construct, CKAR48,49. Because AFs activate PKC in a variety of cell types29,30,31,50, we hypothesized that the mechanism of activation was not cell-type dependent. As primary sinonasal ALIs are very difficult to transfect, even with viral systems, CKAR was transfected into A549 cells, a commonly used lung epithelial cell line. CKAR contains the FHA2 domain of RAD53p as well as a PKC phosphorylation sequence designed to be phosphorylated by all PKC isoforms. These sequences are flanked by an eCFP the N-terminus and a citrine YFP variant on the C-terminus. When phosphorylated, the substrate sequence binds the FHA2 phospho-peptide-binding domain, resulting in a conformational change that keeps the CFP and YFP further apart, reducing FRET emission. Thus, a decrease in FRET emission correlates with an increase in PKC activity, and vice verse. This conformational change is reversible by phosphatases. Single transfected cells were imaged by conventional wide-field low-light-level microscopy, collecting light at three wavelengths: 1) CFP excitation, CFP emission, 2) CFP excitation, YFP emission, and 3) YFP excitation, YFP emission. Data are reported as the signal of wavelength 2 divided by wavelength 1 (i.e. the yellow/cyan emission ratio at cyan excitation, or FRET/CFP ratio) as previously described48.
Application of 10 μM AFB2 caused a decrease in CKAR FRET/CFP ratio that was reversible by addition of Gö6983 in the continued presence of AFB2 (Fig. 7a–c). As a control, A549 CKAR-transfected cells treated with PMA exhibited a fast decrease in CKR FRET that was reversed with application of Gö6983 (1 μM) in the continued presence of PMA (Fig. 7c). Application of forskolin, an activator of adenylate cyclase, had no effect on CKAR fluorescence, as previously described48 (Fig. 7c). These results strongly support the hypothesis that AFB2 exposure increases PKC activity.
The average person inhales hundreds to thousands of airborne Aspergillus spores daily10. In immune-competent individuals, these fungi are typically cleared without consequence. However, in individuals with impaired respiratory defenses (e.g., patients with CRS, diabetes, CF or otherwise immunocompromised), fungal infection can be a significant or even a fatal complication9. Understanding the effects of mycotoxins on the respiratory epithelium is important for understanding the pathogenesis of respiratory (upper and lower) aspergillosis. Here, we show that a class of Aspergillus mycotoxins, aflatoxins, can slow basal and stimulated respiratory CBF, potentially enhancing A. flavus pathogenesis by impairing MCC. Aflatoxins target PKC, previously shown in other studies to slow CBF33. This occurs without alteration of calcium or baseline NO signaling.
The affects observed here are in response to acute aflatoxin exposure. Longer-term studies of the effects of aflatoxins on mucociliary transport, potentially in an animal model of aflatoxin-exposure, will help to shed light on situations of chronic exposure and effects on airways. To our knowledge, studies of longer term exposures have only been previously done in airway cells34,51,52,53,54,55,56,57 and animals28,58,59,60,61,62 with a focus on carcinogenesis. The concentrations of aflatoxins used here (0.1–10 μM) are in the same range used in previous in vitro and ex vivo studies by other groups4,52,53,56,57. However, it must also be determined how environmental aflatoxin exposure, often measured in ppm of aflatoxin-contaminated dust, actually translates to concentrations seen by the airway epithelial cells. As exposure often occurs through contaminated dust, airway deposition will be affected by particle size and sinonasal airflow patterns63. This would be further confounded by the fact that the most commonly used animal models, such as mice, have significantly different paranasal sinus anatomy than humans64,65. Sampling of airway surface liquid and mucus from patients with respiratory A. flavus infections may shed light on concentrations of aflatoxins generated during active A. flavus infection. Moreover, since a significant amount of aflatoxin contamination occurs in grain-based livestock and pet foods66,67,68, inhalation of aflatoxin-contaminated dust may also be a contributor to respiratory infection in non-human animals as well. Antibiotic use in animals is a major driver for the emergence of resistant pathogenic microorganisms69. Further studies of aflatoxin exposure levels in at-risk humans and animal models are critically needed to help complete our understanding of the consequences of both acute and chronic aflatoxin respiratory exposure in humans an animals.
Coupled with previous data that Aspergillus gliotoxin, fumagillin, and helvoilic acid17,18 slow CBF, the current data emphasize that Aspergillus have evolved an armament of mycotoxins to impair MCC and reduce host innate defense. Our data also show that A. niger and A. fumigatus, which cannot secrete aflatoxins, nevertheless secrete mycotoxins that also activate PKC. Certain PKC isoforms have also been linked to inflammation70 and apoptosis71, and thus chronic exposure to aflatoxins and other Aspergillus mycoctoxins may stimulate these processes, exacerbating epithelial damage. PKC inhibitors have been proposed as therapeutics for inflammatory diseases70. Our study suggests that PKC inhibitors may also have potential for fungal airway diseases by relieving mycotoxin-induced repression of ciliary beating. Moreover, the ability of AFB2 to impair sinonasal NO production in response to bacterial AHL-stimulation of T2R receptors suggests that aflatoxins may play an important role in the generation of mixed fungal and bacterial biofilms sometimes observed in airway diseases72. Because we have shown that reduced T2R38 function correlates with gram-negative bacterial infection43, risk of chronic rhinosinusitis73,74, and surgical outcomes in non-polypoid chronic rhinosinusitis75, exposure to aflatoxins and resulting reduction in downstream components of the T2R38 pathway may have important implications for all of these clinical parameters. Moreover, the ability of aflatoxins to impair ciliary activity may have likewise profound clinical consequences during pulmonary aspergillosis caused by A. flavus76.
In conclusion, exposure of ciliated respiratory epithelial cells to AFB2 resulted in a decrease in both baseline and stimulated CBF through calcium-independent activation of PKC. AFB2 also impaired sinonasal epithelial cell bitter taste receptor-driven NO innate immune responses to gram-negative bacterial quorum sensing molecules. These results suggest that aflatoxins may impair MCC and other innate defense pathways, enhancing the pathogenicity of A. flavus and possibly other co-infecting pathogens as well. In addition to their anti-inflammatory effects, PKC inhibitors may be potential therapeutics for fungal respiratory diseases due to their ability to counteract mycotoxin-induced decreases in ciliary beating and MCC.
Materials and Methods
All experimental protocols were reviewed and approved by the Research and Development Committee at the Philadelphia Veterans Affairs Medical Center and were carried out in accordance with both The University of Pennsylvania and The Philadelphia VA Medical Center guidelines regarding use of residual clinical material in research.
Reagents and solutions
Unless indicated, all reagents and solutions were as previously described40,43,77,78. Fluo-4 and DAF-FM were from Invitrogen (Grand Island, NY). Aflatoxins B1 (AFB1) and B2 (AFB2) were from Cayman (Ann Arbor, MI). Gö6983, BIIE-0246, [Leu31,Pro34]-NPY, NPY-(16–36), antagonist G, and calphostin C were from Tocris (Minneapolis, MN USA). All other reagents were from Sigma-Aldrich (St. Louis, MO USA). Stock solutions of aflatoxin were 10 mM in DMSO. Working solutions (10, 1, and 0.1 μM) contained 0.1%, 0.01%, and 0.001% DMSO, respectively, and were made up immediately before use; activities of aqueous solutions were observed to be markedly reduced after ~1–2 hr at room temp. Anti-aflatoxin antibodies (recognizing AFB1, AFB2, and AFG) were from Thermo Scientific (MA-7386) and Sigma Aldrich (A9555).
Generation of sinonasal ALI cultures
Patients undergoing sinonasal surgery were recruited from the Department of Otorhinolaryngology at the University of Pennsylvania and the Philadelphia Veterans Affairs Medical Center with full approval of both Institutional Review Boards (Penn#800614, PVAMC#00781) and written informed consent was obtained for all participating patients in accordance with the U.S. Department of Health and Human Services code of federal regulation Title 45 CFR 46.116. Exclusion criteria included a history of systemic diseases (e.g., Wegner’s, Sarcoid, CF), immunodeficiences, or use of antibiotics, oral corticosteroids, or anti-biologics (e.g. Xolair) within one month of surgery. Human sinonasal epithelial cells were enzymatically dissociated grown to confluence in proliferation medium (DMEM/Ham’s F-12 plus BEBM; Clonetics, Cambrex, East, NJ, USA) for 7 days as previously described37,43. Confluent cells were dissociated and seeded on porous polyester membranes coated with BSA, type I bovine collagen, and fibronectin in cell culture inserts in LHC basal medium (Invitrogen). Culture medium was removed from the upper compartment and basolateral media was changed to differentiation medium (1:1 DMEM:BEBM) containing hEGF (0.5 ng/ ml), epinephrine (5 g/ml), BPE (0.13 mg/ml), hydrocortisone (0.5 g/ml), insulin (5 g/ml), triiodothyronine (6.5 g/ml), and transferrin (0.5 g/ml), supplemented with 100 U/ml penicillin, 100 g/ml streptomycin, 0.1 nM retinoic acid, and NuSerum (BD Biosciences, San Jose, CA) as previously described37,43.
Measurement of ciliary beat frequency (CBF)
Whole-field CBF was measured using the Sisson-Ammons Video Analysis system79 as previously described36,40,43 at ~28–30 °C. Cultures were imaged using at 100 frames/second using a Leica Microscope (20x/0.8NA objective) with Hoffman modulation contrast. Experiments utilized Dulbecco’s PBS (1.8 mM calcium) on the apical side and HEPES-buffered Hank’s Balanced Salt Solution supplemented with 1× MEM vitamins and amino acids on the basolateral side.
Calcium and nitric oxide (NO) imaging
Calcium and NO were imaged using the Fluo-4 and DAF-FM, respectively, as previously described36,43,77,78. Cultures were loaded with Fluo-4 AM (10 μM applied apically) for 2 hrs followed by washing and 20 min incubation in the dark. Cultures were similarly loaded with 10 μM DAF-FM diacetate for 90 min in the presence of 5 μM carboxy-PTIO, followed by washing to remove unloaded DAF-FM and cPTIO and incubation for 15 minutes prior to imaging. Imaging was performed using an Olympus Fluoview confocal system with IX-81 microscope and 10x (0.3 NA UPlanFLN) objective. Images were analyzed using Fluoview software as previously described36,43,77,78. Fluo-4 fluorescence was normalized after subtraction of background, estimated by unloaded ALIs at identical settings. Baseline fluorescence (Fo) was determined from the first 10 frames of each experiment. DAF-FM measurements utilized raw fluorescence values to compare experiments performed under identical conditions and settings.
Cultures of A. niger (strain WB326 [ATCC16888] and a clinical isolate from the Philadelphia VA Medical Center), A. fumigatus (NIH5233 [ATCC 13073] and NRRL163 [ATCC1022]), and A. flavus (NRRL3357 [ATCC200026]) were grown in 40 ml BACTEC Myco/F Lytic Culture Vials (BD, Sparks, MD) with assistance from the Philadelphia VA clinical microbiology lab. Inoculated cultures were grown at 30 °C for ten days. Conditioned medium (CM) was extracted and filtered sequentially through 0.45 μm and 0.2 μm filters.
A549 cell culture, transfection, and CKAR FRET imaging
A549 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA) and cultured in Kaighn’s modification of Ham’s F12 media (F12K) with 10% fetal bovine serum and 1x penicillin-streptomycin mix (Gibco/Thermo Fisher Scientific, Waltham, MA). Cells were used at passage 15–20. Cells were transfected with CKAR48,49 (Alexandra Newton, University of California San Diego, Addgene, Cambridge, MA, plasmid#14860) by standard calcium phosphate transfection in a 150 mm dish at ~75% confluency. The day after transfection, cells were trypsinized and re-plated into chambered coverglass wells (CellVis, Mountain View, CA) at 50% confluency. Cells were used at 48 hrs after transfection, and cells media was replaced with 10 mM HEPES-buffered Hank’s balanced salt solution (HBSS) and imaging was performed at room temperature on the stage of an Olympus IX-83 inverted microscope (60x PlanApo 1.4 NA oil-immersion objective; Olympus Life Sciences, Tokyo, Japan) equipped with excitation and emission filter wheels (Sutter Instruments, Novato, CA) and a CFP-YFP FRET filter set (89003-ET, Chroma Technologies, Rockingham, VT). Images were acquired (12 sec intervals) and analyzed using MetaFluor (Molecular Devices, Sunnyvale, CA) and ratio images were constructed using ImageJ (W. Rasband, National Institutes of Mental Health, Research Services Branch, Bethesda, MD). Both background (estimated using an off-cell area) and baseline drift were subtracted as described49 before averaging of traces.
Data analysis and statistics
One-way analysis of variance (ANOVA) was performed in GraphPad Prism with appropriate post-tests as indicated; P < 0.05 was considered statistically significant. All other data analysis was performed in Excel. For all figures, one (*) and two (**) asterisks indicate P < 0.05 and P < 0.01 respectively; “n.s.” indicates no statistical significance. All data are mean ± SEM.
How to cite this article: Lee, R. J. et al. Fungal Aflatoxins Reduce Respiratory Mucosal Ciliary Function. Sci. Rep. 6, 33221; doi: 10.1038/srep33221 (2016).
Antunes, M. B., Gudis, D. A. & Cohen, N. A. Epithelium, cilia, and mucus: their importance in chronic rhinosinusitis. Immunol Allergy Clin North Am 29, 631–643 (2009).
Bhattacharyya, N., Grebner, J. & Martinson, N. G. Recurrent acute rhinosinusitis: epidemiology and health care cost burden. Otolaryngology–head and neck surgery: official journal of American Academy of Otolaryngology-Head and Neck Surgery 146, 307–312 (2012).
Blackwell, D. L., Collins, J. G. & Coles, R. Summary health statistics for U.S. adults: National Health Interview Survey, 1997. Vital Health Stat 10, 1–109 (2002).
Van Vleet, T. R., Mace, K. & Coulombe, R. A. Jr. Comparative aflatoxin B(1) activation and cytotoxicity in human bronchial cells expressing cytochromes P450 1A2 and 3A4. Cancer research 62, 105–112 (2002).
Cherry, D. K. & Woodwell, D. A. National Ambulatory Medical Care Survey: 2000 summary. Adv Data 1–32 (2002).
Marcinkiewicz, J., Strus, M. & Pasich, E. Antibiotic resistance: a “dark side” of biofilmassociated chronic infections. Polskie Archiwum Medycyny Wewnetrznej 123, 309–313 (2013).
Manes, R. P. & Batra, P. S. Bacteriology and antibiotic resistance in chronic rhinosinusitis. Facial plastic surgery clinics of North America 20, 87–91 (2012).
Hens, G. & Hellings, P. W. The nose: gatekeeper and trigger of bronchial disease. Rhinology 44, 179–187 (2006).
Soler, Z. M. & Schlosser, R. J. The role of fungi in diseases of the nose and sinuses. American journal of rhinology & allergy 26, 351–358 (2012).
Hohl, T. M. & Feldmesser, M. Aspergillus fumigatus: principles of pathogenesis and host defense. Eukaryotic cell 6, 1953–1963 (2007).
Chaudhary, N. & Marr, K. A. Impact of Aspergillus fumigatus in allergic airway diseases. Clinical and translational allergy 1, 4 (2011).
Hutcheson, P. S., Schubert, M. S. & Slavin, R. G. Distinctions between allergic fungal rhinosinusitis and chronic rhinosinusitis. American journal of rhinology & allergy 24, 405–408 (2010).
Amitani, R. et al. Effects of human neutrophil elastase and Pseudomonas aeruginosa proteinases on human respiratory epithelium. Am J Respir Cell Mol Biol 4, 26–32 (1991).
Kanthakumar, K. et al. Mechanisms of action of Pseudomonas aeruginosa pyocyanin on human ciliary beat in vitro. Infection and immunity 61, 2848–2853 (1993).
Read, R. C. et al. Effect of Pseudomonas aeruginosa rhamnolipids on mucociliary transport and ciliary beating. J Appl Physiol 72, 2271–2277 (1992).
Steinfort, C. et al. Effect of Streptococcus pneumoniae on human respiratory epithelium in vitro. Infection and immunity 57, 2006–2013 (1989).
Amitani, R. et al. Aspergillus culture filtrates and sputum sols from patients with pulmonary aspergillosis cause damage to human respiratory ciliated epithelium in vitro. Eur Respir J 8, 1681–1687 (1995).
Amitani, R. et al. Purification and characterization of factors produced by Aspergillus fumigatus which affect human ciliated respiratory epithelium. Infection and immunity 63, 3266–3271 (1995).
Abad, A. et al. What makes Aspergillus fumigatus a successful pathogen? Genes and molecules involved in invasive aspergillosis. Revista iberoamericana de micologia 27, 155–182 (2010).
Krishnan, S., Manavathu, E. K. & Chandrasekar, P. H. Aspergillus flavus: an emerging non-fumigatus Aspergillus species of significance. Mycoses 52, 206–222 (2009).
Pasqualotto, A. C. Differences in pathogenicity and clinical syndromes due to Aspergillus fumigatus and Aspergillus flavus. Medical mycology: official publication of the International Society for Human and Animal Mycology 407 Suppl 1, S261–S270 (2009).
Sutandyo, N. Nutritional carcinogenesis. Acta medica Indonesiana 42, 36–42 (2010).
Bedard, L. L. & Massey, T. E. Aflatoxin B1-induced DNA damage and its repair. Cancer letters 241, 174–183 (2006).
Olsen, J. H., Dragsted, L. & Autrup, H. Cancer risk and occupational exposure to aflatoxins in Denmark. British journal of cancer 58, 392–396 (1988).
Shen, H. et al. Aflatoxin G1-induced oxidative stress causes DNA damage and triggers apoptosis through MAPK signaling pathway in A549 cells. Food and chemical toxicology: an international journal published for the British Industrial Biological Research Association 62, 661–669 (2013).
Kelly, J. D., Eaton, D. L., Guengerich, F. P. & Coulombe, R. A. Jr. Aflatoxin B1 activation in human lung. Toxicology and applied pharmacology 144, 88–95 (1997).
Dvorackova, I., Stora, C. & Ayraud, N. Evidence of aflatoxin B1 in two cases of lung cancer in man. Journal of cancer research and clinical oncology 100, 221–224 (1981).
Massey, T. E., Smith, G. B. & Tam, A. S. Mechanisms of aflatoxin B1 lung tumorigenesis. Experimental lung research 26, 673–683 (2000).
Mistry, K. J., Krishna, M. & Bhattacharya, R. K. Modulation of aflatoxin B1 activated protein kinase C by phenolic compounds. Cancer letters 121, 99–104 (1997).
Mistry, K. J., Krishna, M., Pasupathy, K., Murthy, V. & Bhattacharya, R. K. Signal transduction mechanism in response to aflatoxin B1 exposure: protein kinase C activity. Chemico-biological interactions 100, 177–185 (1996).
Van den Heever, L. H. & Dirr, H. W. Effect of aflatoxin B1 on human platelet protein kinase C. The International journal of biochemistry 23, 839–843 (1991).
Salathe, M. Regulation of mammalian ciliary beating. Annual review of physiology 69, 401–422 (2007).
Salathe, M., Pratt, M. M. & Wanner, A. Protein kinase C-dependent phosphorylation of a ciliary membrane protein and inhibition of ciliary beating. Journal of cell science 106 (Pt 4), 1211–1220 (1993).
Wong, J. J. & Hsieh, D. P. Mutagenicity of aflatoxins related to their metabolism and carcinogenic potential. Proceedings of the National Academy of Sciences of the United States of America 73, 2241–2244 (1976).
Butler, W. H., Greenblatt, M. & Lijinsky, W. Carcinogenesis in rats by aflatoxins B1, G1, and B2. Cancer research 29, 2206–2211 (1969).
Zhao, K. Q. et al. Molecular modulation of airway epithelial ciliary response to sneezing. FASEB journal: official publication of the Federation of American Societies for Experimental Biology 26, 3178–3187 (2012).
Lai, Y. et al. Inflammation-mediated upregulation of centrosomal protein 110, a negative modulator of ciliogenesis, in patients with chronic rhinosinusitis. The Journal of allergy and clinical immunology 128, 1207–1215 e1201 (2011).
Wu-Zhang, A. X. & Newton, A. C. Protein kinase C pharmacology: refining the toolbox. Biochem J 452, 195–209 (2013).
Lee, R. J. & Foskett, J. K. Ca2+ signaling and fluid secretion by secretory cells of the airway epithelium. Cell calcium 55, 325–336 (2014).
Lee, R. J. et al. Vasoactive intestinal peptide regulates sinonasal mucociliary clearance and synergizes with histamine in stimulating sinonasal fluid secretion. FASEB journal: official publication of the Federation of American Societies for Experimental Biology 27, 5094–5103 (2013).
Wong, L. B., Park, C. L. & Yeates, D. B. Neuropeptide Y inhibits ciliary beat frequency in human ciliated cells via nPKC, independently of PKA. Am J Physiol 275, C440–C448 (1998).
Jones, D. A., Cummings, J., Langdon, S. P. & Smyth, J. F. Preclinical studies on the broad-spectrum neuropeptide growth factor antagonist G. General pharmacology 28, 183–189 (1997).
Lee, R. J. et al. T2R38 taste receptor polymorphisms underlie susceptibility to upper respiratory infection. The Journal of clinical investigation 122, 4145–4159 (2012).
Lee, R. J. & Cohen, N. A. Taste receptors in innate immunity. Cellular and molecular life sciences: CMLS 72, 217–236 (2015).
Lee, R. J. & Cohen, N. A. Role of the bitter taste receptor T2R38 in upper respiratory infection and chronic rhinosinusitis. Curr Opin Allergy Clin Immunol 15, 14–20 (2015).
Michell, B. J. et al. Coordinated control of endothelial nitric-oxide synthase phosphorylation by protein kinase C and the cAMP-dependent protein kinase. The Journal of biological chemistry 276, 17625–17628 (2001).
Matsubara, M., Hayashi, N., Jing, T. & Titani, K. Regulation of endothelial nitric oxide synthase by protein kinase C. Journal of biochemistry 133, 773–781 (2003).
Violin, J. D., Zhang, J., Tsien, R. Y. & Newton, A. C. A genetically encoded fluorescent reporter reveals oscillatory phosphorylation by protein kinase C. The Journal of cell biology 161, 899–909 (2003).
Gallegos, L. L. & Newton, A. C. Genetically encoded fluorescent reporters to visualize protein kinase C activation in live cells. Methods Mol Biol 756, 295–310 (2011).
Mistry, K. J., Krishna, M. & Bhattacharya, R. K. Effect of aflatoxin B1 on phosphoinositide signal transduction pathway during regeneration of liver cells following partial hepatectomy. Indian journal of biochemistry & biophysics 38, 270–273 (2001).
Zhang, Z. et al. Cytochrome P450 2A13 mediates the neoplastic transformation of human bronchial epithelial cells at a low concentration of aflatoxin B1. Int J Cancer 134, 1539–1548 (2014).
Van Vleet, T. R., Watterson, T. L., Klein, P. J. & Coulombe, R. A. Jr. Aflatoxin B1 alters the expression of p53 in cytochrome P450-expressing human lung cells. Toxicol Sci 89, 399–407 (2006).
Van Vleet, T. R., Klein, P. J. & Coulombe, R. A. Jr. Metabolism of aflatoxin B1 by normal human bronchial epithelial cells. J Toxicol Environ Health A 63, 525–540 (2001).
Paget, V., Sichel, F., Garon, D. & Lechevrel, M. Aflatoxin B1-induced TP53 mutational pattern in normal human cells using the FASAY (Functional Analysis of Separated Alleles in Yeast). Mutat Res 656, 55–61 (2008).
Lorico, A., Nesland, J., Emilsen, E., Fodstad, O. & Rappa, G. Role of the multidrug resistance protein 1 gene in the carcinogenicity of aflatoxin B1: investigations using mrp1-null mice. Toxicology 171, 201–205 (2002).
He, X. Y. et al. Efficient activation of aflatoxin B1 by cytochrome P450 2A13, an enzyme predominantly expressed in human respiratory tract. Int J Cancer 118, 2665–2671 (2006).
Cui, A. et al. Aflatoxin B1 induces Src phosphorylation and stimulates lung cancer cell migration. Tumour Biol 36, 6507–6513 (2015).
Mulder, J. E., Bondy, G. S., Mehta, R. & Massey, T. E. The impact of chronic Aflatoxin B1 exposure and p53 genotype on base excision repair in mouse lung and liver. Mutat Res 773, 63–68 (2015).
Mulder, J. E., Bondy, G. S., Mehta, R. & Massey, T. E. Up-regulation of nucleotide excision repair in mouse lung and liver following chronic exposure to aflatoxin B(1) and its dependence on p53 genotype. Toxicology and applied pharmacology 275, 96–103 (2014).
Larsson, P., Persson, E., Tyden, E. & Tjalve, H. Cell-specific activation of aflatoxin B1 correlates with presence of some cytochrome P450 enzymes in olfactory and respiratory tissues in horse. Res Vet Sci 74, 227–233 (2003).
Harvey, R. B., Edrington, T. S., Kubena, L. F., Elissalde, M. H. & Rottinghaus, G. E. Influence of aflatoxin and fumonisin B1-containing culture material on growing barrows. Am J Vet Res 56, 1668–1672 (1995).
Dilkin, P. et al. Toxicological effects of chronic low doses of aflatoxin B(1) and fumonisin B(1)-containing Fusarium moniliforme culture material in weaned piglets. Food and chemical toxicology: an international journal published for the British Industrial Biological Research Association 41, 1345–1353 (2003).
Schwab, J. A. & Zenkel, M. Filtration of particulates in the human nose. The Laryngoscope 108, 120–124 (1998).
Jacob, A. & Chole, R. A. Survey anatomy of the paranasal sinuses in the normal mouse. The Laryngoscope 116, 558–563 (2006).
Dalgorf, D. M. & Harvey, R. J. Chapter 1: Sinonasal anatomy and function. American journal of rhinology & allergy 27 Suppl 1, S3–S6 (2013).
Streit, E. et al. Current situation of mycotoxin contamination and co-occurrence in animal feed–focus on Europe. Toxins (Basel) 4, 788–809 (2012).
Streit, E., Naehrer, K., Rodrigues, I. & Schatzmayr, G. Mycotoxin occurrence in feed and feed raw materials worldwide: long-term analysis with special focus on Europe and Asia. J Sci Food Agric 93, 2892–2899 (2013).
Leung, M. C., Diaz-Llano, G. & Smith, T. K. Mycotoxins in pet food: a review on worldwide prevalence and preventative strategies. J Agric Food Chem 54, 9623–9635 (2006).
Economou, V. & Gousia, P. Agriculture and food animals as a source of antimicrobial-resistant bacteria. Infect Drug Resist 8, 49–61 (2015).
Diaz-Meco, M. T. & Moscat, J. The atypical PKCs in inflammation: NF-kappaB and beyond. Immunological reviews 246, 154–167 (2012).
Zhao, M., Xia, L. & Chen, G. Q. Protein kinase cdelta in apoptosis: a brief overview. Archivum immunologiae et therapiae experimentalis 60, 361–372 (2012).
Cohen, M. et al. Biofilms in chronic rhinosinusitis: a review. American journal of rhinology & allergy 23, 255–260 (2009).
Adappa, N. D. et al. The bitter taste receptor T2R38 is an independent risk factor for chronic rhinosinusitis requiring sinus surgery. International forum of allergy & rhinology 4, 3–7 (2014).
Adappa, N. D. et al. Genetics of the taste receptor T2R38 correlates with chronic rhinosinusitis necessitating surgical intervention. International forum of allergy & rhinology 3, 184–187 (2013).
Adappa, N. D. et al. TAS2R38 genotype predicts surgical outcome in nonpolypoid chronic rhinosinusitis. International forum of allergy & rhinology (2015).
Mahgoub, E. S. & el-Hassan, A. M. Pulmonary aspergillosis caused by Aspergillus flavus. Thorax 27, 33–37 (1972).
Lee, R. J. et al. Bitter and sweet taste receptors regulate human upper respiratory innate immunity. The Journal of clinical investigation 124, 1393–1405 (2014).
Lee, R. J., Chen, B., Redding, K. M., Margolskee, R. F. & Cohen, N. A. Mouse nasal epithelial innate immune responses to Pseudomonas aeruginosa quorum-sensing molecules require taste signaling components. Innate immunity 20, 606–617 (2014).
Sisson, J. H., Stoner, J. A., Ammons, B. A. & Wyatt, T. A. All-digital image capture and whole-field analysis of ciliary beat frequency. Journal of microscopy 211, 103–111 (2003).
The authors wish to thank Dr. L. Chandler and the Philadelphia V.A. Medical Center microbiology laboratory for help with Aspergillus culture. This study was supported by grants from National Institutes of Health (R01DC013588 to N.A.C., R03DC013862 to R.J.L.), a charitable donation from the RLG Foundation, Inc (to N.A.C.), and funding from the Department of Otorhinolaryngology at the University of Pennsylvania (to R.J.L.).
The authors declare no competing financial interests.
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Lee, R., Workman, A., Carey, R. et al. Fungal Aflatoxins Reduce Respiratory Mucosal Ciliary Function. Sci Rep 6, 33221 (2016). https://doi.org/10.1038/srep33221
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