Ribonuclease T2 from Aspergillus fumigatus promotes T helper type 2 responses through M2 polarization of macrophages

Abstract

Allergic bronchopulmonary aspergillosis (ABPA) is an allergic immunological response to Aspergillus 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 from Af (Af RNASET2) induces M2‑type macrophage polarization to produce a T helper 2 (Th2) immune response. Recombinant Af RNASET2 (rAf RNASET2) was expressed and purified in a prokaryotic pET system and BALB/c mice were immunized with rAf RNASET2 for in vivo analyses. Expression levels of M2 polarization factors were evaluated in RAW264.7 macrophages treated with rAf RNASET2 in vitro using flow cytometry, reverse transcription‑quantitative PCR, and western blot analysis. The results predicted that the mature Af RNASET2 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 rAf RNASET2. RAW264.7 macrophages treated with rAf RNASET2 showed increased mRNA expression levels of M2 factors [arginase 1, Il‑10, and Il‑13]; however, there was no difference in cells treated with rAf RNASET2 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 rAf RNASET2. In addition, rAf RNASET2 upregulated the expression of phosphorylated mitogen activated protein kinases (MAPKs) in RAW264.7 cells, whereas MAPK inhibitors attenuated rAf RNASET2‑induced IL‑10 expression in RAW264.7 cells. In conclusion, the present study reveals that high rAf RNASET2 activity is required for rAf RNASET2‑induced M2 polarization of macrophages and suggests an important immune regulatory role for Af RNASET2 in ABPA pathogenesis.

Figure 1

Amino acid sequence homology of Af RNASET2, Sj CP1412, Sm ω-1, and Cs RNASET2. Common amino acids are indicated by the grey shading. Black solid lines indicate conserved functional domains, CAS-1 and CAS-2. Solid black triangles indicate conserved cysteine residues. Af, Aspergillus fumigatus; Sj, Schistosoma japonicum; Sm, Schistosoma mansoni; Cs, Clonorchis sinensis; CAS, conserved amino acid sequences; RNASET2, T2 ribonuclease.

Figure 2

Expression, purification, and RNase activity of rAf RNASET2. (A) Analysis of the prokaryotic recombinant expression vector pET-His-Af RNASET2 (without signal peptides, Glutamic acid 26-Arginine 407) by double enzyme digestion. Lane M indicates the DNA marker. The 1,200 bp band indicates the Af RNASET2 cDNA insert. (B) SDS-PAGE analysis of pET-His-Af RNASET2 expression. Lane M, protein marker; lane 1, E. coli transformed with pET-His-Af RNASET2 prior to IPTG induction; lane 2, E. coli transformed with pET-His-Af RNASET2 following IPTG induction; lane 3, sediment from E. coli transformed with pET-His-Af RNASET2 plasmid following IPTG induction, and ultrasonication. (C) SDS-PAGE of renatured rAf RNASET2 protein following purification using Ni-NTA affinity chromatography. Lane M indicates the protein marker. (D) Analysis of RNase activity of rAf RNASET2 (1 µg) with yeast RNA (50 µg) at 37°C at 20, 40, 60, 80 and 100 min). (E) Analysis of RNase activity of rAf RNASET2 with or without RNasin (RNAse inhibitor) pretreatment. RNase enzymolysis products were analyzed using 1% agarose gel electrophoresis. E. coli, Escherichia coli; r, recombinant; Af, Aspergillus fumigatus; RNASET2, T2 ribonuclease; IPTG, isopropyl-β-d-thiogalactopyranoside.

Figure 3

In vivo induction of immune responses by rAf RNASET2. Serum obtained from orbital vein blood (collected 7 days after the third immunization day) from BABL/c mice injected with rAf RNASET2 with aluminum adjuvant, aluminum adjuvant alone, or PBS only (n=6 per group) were analyzed using ELISA. Levels of (A) IL-4, (B) IL-10, (C) IL-13 and (D) INF-γ were increased in the rAf RNASET2 with aluminum adjuvant immunization group compared with that in the control groups. Data are presented as the mean ± SD from independent experiments. ^**P<0.01. r, recombinant; Af, Aspergillus fumigatus; RNASET2, T2 ribonuclease.

Figure 4

Macrophage polarization effect of rAf RNASET2. Flow cytometry of (A) CD16/32 and (B) CD206 expression levels on the surface of RAW264.7 cells stimulated with 20 or 40 µg/ml rAf RNASET2. Representative images from one of three independent experiments are shown. Data are presented as mean ± SD. ^**P<0.01. r, recombinant; Af, Aspergillus fumigatus; RNASET2, T2 ribonuclease.

Figure 5

rAf RNASET2 increases expression of M2 polarization markers. Reverse transcription-quantitative PCR and western blot analysis were conducted with RAW264.7 macrophages co-cultured with rAf RNASET2 (20 µg/ml), inactive rAf RNASET2 (pretreated with RNasin), and PBS and LPS controls at 37°C with 5% CO₂ for 24 h. mRNA expression levels of (A) Arg-1, (B) iNos, (C) IL-10, and (D) IL-13. Data are presented as mean ± SD from 3 independent experiments. ^**P<0.01. (E) Western blot analysis of ARG-1, COX2, iNOS, IL-10, and GAPDH in RAW264.7 macrophages following rAf RNASET2 (20 µg/ml) treatment for 24 h. r, recombinant; Af, Aspergillus fumigatus; RNASET2, T2 ribonuclease; LPS, lipopolysaccharide; iNOS, induced nitric oxide synthase; IL, interleukin; Arg-1, arginase-1.

Figure 6

rAf RNASET2 upregulates proteins in the MAPK signaling pathway in macrophages. (A) Western blot analysis of JNK, p-JNK, p38, p-p38, ERK1/2, and p-ERK1/2 in RAW264.7 macrophages following treatment with rAf RNASET2 (20 µg/ml) or (24-h) RNasin-inactivated rAf RNASET2. Levels of IL-10 in RAW264.7 macrophages treated with rAf RNASET2 (20 µg/ml) alone or in the presence of MAPK signal inhibitors (U0126, SP600125 and SB203580 for ERK, JNK and p38, respectively) were determined using (B) reverse transcription-quantitative PCR and (C) ELISA. Data are represented as mean ± SD from 3 independent experiments. ^**P<0.01. r, recombinant; Af, Aspergillus fumigatus; RNASET2, T2 ribonuclease; p, phosphorylated; LPS, lipopolysaccharide.