Fungal Aflatoxins Reduce Respiratory Mucosal Ciliary Function

Abstract

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.

Protease‐activated receptor 2 activates airway apical membrane chloride permeability and increases ciliary beating

“…To determine whether PAR-2 Ca 2+ signals had an effect on CBF, we imaged CBF in differentiated, primary, sinonasal ALIs, which form functional motile cilia ( Fig. 6A), as previously described (30,34,38). 2FLI induced a transient increase in CBF (Fig.…”

“…We previously observed that histamine elicits Ca 2+ responses from primary sinonasal epithelial cells that are sufficient (at high histamine concentrations) to elicit Ca 2+ -activated fluid secretion but not increase CBF (30). Moreover, because PARs can activate PKC (26,(31)(32)(33), which can slow CBF (34), and b-arrestindependent pathways (32,35), PARs could have a range of effects on CBF. To our knowledge, no one has yet investigated CBF responses to PAR-2 stimulation in primary differentiated human airway cells.…”

Inverse regulation of secretion and inflammation in human airway gland serous cells by neuropeptides upregulated in allergy and asthma

“…Coupled with increased inflammation in the presence of NPY, our data suggest NPY may have multiple detrimental effects in diseases of mucus thickening/mucostasis. We (129) and others (130)(131)(132) have shown that NPY decreases airway ciliary beat frequency, which may further impair mucociliary clearance.…”

“…Isolation of primary serous acinar cells, immunofluorescence, and live cell imaging of acinar cell volume, pHi (SNARF-5F), and Cl -(SPQ) was carried out as described (10-12, 47, 48). ASL height and pH measurements and ELISAs were carried out as previously reported (92,129,(151)(152)(153)(154)(155)(156)(157). Bacterial growth assays were carried out as previously described (158,159).…”

Altered polarization of PAR-2 signaling during airway epithelial remodeling

“…8,22,23 Fungal culture was carried out as described. 24 Imaging of ASL height was performed as described. 25 FITC-conjugated dextran permeability was measured as described.…”