Role of Aspergillus fumigatus in Triggering Protease-Activated Receptor-2 in Airway Epithelial Cells and Skewing the Cells toward a T-helper 2 Bias.

Aspergillus fumigatus (AF) infection and sensitization are common and promote Th2 disease in individuals with asthma. Innate immune responses of bronchial epithelial cells are now known to play a key role in determination of T cell responses upon encounter with inhaled pathogens. We have recently shown that extracts of AF suppress JAK-STAT signaling in epithelial cells and thus may promote Th2 bias. To elucidate the impact of AF on human bronchial epithelial cells, we tested the hypothesis that AF can modulate the response of airway epithelial cells to favor a Th2 response and explored the molecular mechanism of the effect. Primary normal human bronchial epithelial (NHBE) cells were treated with AF extract or fractionated AF extract before stimulation with poly I:C or infection with human rhinovirus serotype 16 (HRV16). Expression of CXCL10 mRNA (real-time RT-PCR) and protein (ELISA) were measured as markers of IFN-mediated epithelial Th1-biased responses. Western blot was performed to evaluate expression of IFN regulatory factor-3 (IRF-3), NF-κB, and tyrosine-protein phosphatase nonreceptor type 11 (PTPN11), which are other markers of Th1 skewing. Knockdown experiments for protease-activated receptor-2 (PAR-2) and PTPN11 were performed to analyze the role of PAR-2 in the mechanism of suppression by AF. AF and a high-molecular-weight fraction of AF extract (HMW-AF; > 50 kD) profoundly suppressed poly I:C- and HRV16-induced expression of both CXCL10 mRNA and protein from NHBE cells via a mechanism that relied upon PAR-2 activation. Both AF extract and a specific PAR-2 activator (AC-55541) suppressed the poly I:C activation of phospho-IRF-3 without affecting activation of NF-κB. Furthermore, HMW-AF extract enhanced the expression of PTPN11, a phosphatase known to inhibit IFN signaling, and concurrently suppressed poly I:C-induced expression of both CXCL10 mRNA and protein from NHBE cells. These results show that exposure of bronchial epithelial cells to AF extract suppressed poly I:C and HRV16 signaling via a mechanism shown to involve activation of PAR-2 and PTPN11. This action of AF may promote viral disease exacerbations and may skew epithelial cells to promote Th2 inflammation in allergic airway disorders mediated or exacerbated by AF, such as asthma and chronic rhinosinusitis.

Suppression of epithelial signal transducer and activator of transcription 1 activation by extracts of Aspergillus fumigatus.

Aspergillus fumigatus (AF) is often pathogenic in immune-deficient individuals and can cause life-threatening infections such as invasive aspergillosis. The pulmonary epithelial response to AF infection and the signaling pathways associated with it have not been completely studied. BEAS-2B cells or primary human bronchial epithelial cells were exposed to extracts of AF and challenged with IFN-ß or the Toll-like receptor 3 agonist double-stranded RNA (dsRNA). Cytokine release (B-cell activating factor of the TNF family [BAFF], IFN-γ-induced protein-10 [IP-10], etc.) was assessed. AF extract was separated into low-molecular-weight (LMW) and high-molecular-weight (HMW) fractions using ultra 4 centrifugal force filters to characterize the activity. Real-time PCR was performed with a TaqMan method, and protein estimation was performed using ELISA techniques. Western blot was performed to assess phosphorylation of signal transducer and activator of transcription 1 (STAT1). IFN-ß and dsRNA induced messenger RNA (mRNA) expression of BAFF (350- and 452-fold, respectively [n = 3]) and IP-10 (1,081- and 3,044-fold, respectively [n = 3]) in BEAS-2B cells. When cells were pretreated with AF extract for 1 hour and then stimulated with IFN-ß or dsRNA for 6 hours, induction of BAFF and IP-10 mRNA was strongly suppressed relative to levels produced by IFN-ß and dsRNA alone. When compared with control, soluble BAFF and IP-10 protein levels were maximally suppressed in dsRNA-stimulated wells treated with 1:320 wt/vol AF extract (P < 0.005). Upon molecular size fractionation, a LMW fraction of AF extract had no measurable suppressive effect on IP-10 mRNA expression. However, a HMW fraction of the AF extract significantly suppressed IP-10 expression in BEAS-2B cells that were stimulated with dsRNA or IFN-ß. When BEAS-2B cells were pretreated with AF extract and then stimulated with IFN-ß, reduced levels of pSTAT1 were observed, with maximum suppression at 4 and 6 hours. Our results show that AF extracts suppressed expression of inflammatory cytokines in association with inhibition of the IFN-ß signaling pathway and suppression of the formation of pSTAT1.