Ribonuclease T2 Fromaspergillus Fumigatuspromotes T Helper Type 2 Responses Through M2 Polarization Of Macrophages

Allergic bronchopulmonary aspergillosis (ABPA) is an allergic immunological response toAspergillus fumigatus(Af) exposure, which induces a strong T helper 2 (Th2) response via mechanisms that have yet to be elucidated. The aim of the present study was to investigate the hypothesis that T2 ribonuclease fromAf(AfRNASET2) induces M2-type macrophage polarization to produce a T helper 2 (Th2) immune response. RecombinantAfRNASET2 (rAfRNASET2) was expressed and purified in a prokaryotic pET system and BALB/c mice were immunized with rAfRNASET2 forin vivoanalyses. Expression levels of M2 polarization factors were evaluated in RAW264.7 macrophages treated with rAfRNASET2in vitrousing flow cytometry, reverse transcription-quantitative PCR, and western blot analysis. The results predicted that the matureAfRNASET2 protein (382 amino acids; GenBank no. MN593022) contained two conserved amino acid sequence (CAS) domains, termed CAS-1 and CAS-2, which are also characteristic of the RNASET2 family proteins. The protein expression levels of the Th2-related cytokines interleukin (IL)-4, IL-10, and IL-13 were upregulated in mice immunized with rAfRNASET2. RAW264.7 macrophages treated with rAfRNASET2 showed increased mRNA expression levels of M2 factors [arginase 1,Il-10, andIl-13]; however, there was no difference in cells treated with rAfRNASET2 that had been inactivated with a ribonuclease inhibitor (RNasin). The protein expression levels of IL-10 in macrophage culture supernatant were also increased following stimulation with rAfRNASET2. In addition, rAfRNASET2 upregulated the expression of phosphorylated mitogen activated protein kinases (MAPKs) in RAW264.7 cells, whereas MAPK inhibitors attenuated rAfRNASET2-induced IL-10 expression in RAW264.7 cells. In conclusion, the present study reveals that high rAfRNASET2 activity is required for rAfRNASET2-induced M2 polarization of macrophages and suggests an important immune regulatory role forAfRNASET2 in ABPA pathogenesis.