Suppression of Epithelial Signal Transducer and Activator of Transcription 1 Activation by Extracts of Aspergillus fumigatus

Abstract

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-b or the Toll-like receptor 3 agonist double-stranded RNA (dsRNA). Cytokine release (B-cell activating factor of the TNF family [BAFF], IFN-g-induced protein-10 [IP-10], etc.) was assessed. AF extract was separated into low-molecular-weight (LMW) and highmolecular-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-b 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-b or dsRNA for 6 hours, induction of BAFF and IP-10 mRNA was strongly suppressed relative to levels produced by IFN-b 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-b. When BEAS-2B cells were pretreated with AF extract and then stimulated with IFN-b, 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-b signaling pathway and suppression of the formation of pSTAT1. Clinical RelevanceThe components of Aspergillus fumigatus extract that inhibit JAK-signal transducer and activator of transcription 1 (STAT1) signaling could impair antiviral immunity and could also contribute to the skewing of adaptive immune responses toward T helper (Th) 2 observed in Aspergillus-infected individuals by undermining the Th1 responses via inhibition of the activation of STAT1. We believe that the inhibitory effect on STAT1 activation could be exploited for beneficial effects once we better understand the identity of the factor and the molecular mechanism by which STAT1 phosphorylation is inhibited.

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

“…Recently, we have reported a rapid dephosphorylation of IFN-β-induced phospho-STAT1 triggered by AF extract and have confirmed this in the present study (Supplemental Fig. 2A, B) (6). Since the apparent dephosphorylation of IRF-3 shown in Fig.…”

“…Previous studies by our laboratory and others have shown that extracts of Aspergillus fumigatus suppress Page 13 of 44 activation of epithelial cells by either IFN or by the TLR3 agonist poly I:C (6,18,28,29). In the present study, we show that AF extract also suppressed the HRV16-induced innate immune response in fully differentiated NHBE cells using an air liquid interface culture system (Fig.…”

“…Consistent with this observation, supplemental IFN-β inhalation therapy was effective in treatment of severe asthmatics (42). (6). Since allergic airways diseases are often associated with fungal infections, it is possible that fungi may contribute to the reduced IFN signaling in asthma and CRS.…”

“…Porter et al recently reported that environmental fungi directly contribute to the pathogenesis of chronic Th2-related airway diseases (5). Recently, we have described the inhibition of IFN-β signaling through JAK-STAT1 by Aspergillus fumigatus (AF) in bronchial epithelium, resulting in reduced induction of CXCL10, also known as IFN-γ induced protein 10 (IP-10), a chemokine that attracts Th1 cells (6). Inhibition of IFN signaling by AF in epithelium could bias the epithelial responses toward Th2 chemokines and cytokines and therefore may be important in the mechanism by which AF worsens allergic inflammatory diseases.…”

“…We have noticed changes in cell morphology and viability induced by AF or LMW-AF extract when incubating the cells with concentrated solutions for longer than 24 hours. In contrast, HMW-AF extract did not alter cell morphology or viability (6). Therefore, we utilized HMW-AF extract for experiments that required longer incubations (see below).…”