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

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 M2­type 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 transcription­quantitative 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 CAS­1 and CAS­2, which are also characteristic of the RNASET2 family proteins. The protein expression levels of the Th2­related cytokines interleukin (IL)­4, IL­10, and IL­13 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, Il­10, and Il­13]; 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 IL­10 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 RNASET2­induced IL­10 expression in RAW264.7 cells. In conclusion, the present study reveals that high rAf RNASET2 activity is required for rAf RNASET2­induced M2 polarization of macrophages and suggests an important immune regulatory role for Af RNASET2 in ABPA pathogenesis.

Identification of immunodominant IgE epitopes of the major house dust mite allergen Der f 24.

Previously, a ubiquinol­cytochrome c reductase binding protein (UQCRB) homolog was identified in the house dust mite (HDM) species Dermatophagoides farinae (Der f) as a major allergen. In the present study, the immunodominant immunoglobulin E (IgE) epitope of the protein Der f 24 was investigated. Analysis of the homologous amino acid (aa) sequences in Der f and human UQCRB was performed. Four different recombinant Der f 24 and hybrid proteins formed by integrating Der f and human UQCRB sequences were expressed in Escherichia coli, purified using Ni­NTA resins, and IgE­binding activity was determined using IgE­western blotting and enzyme­linked immunosorbent assay (ELISA) experiments. IgE epitopes were further identified by IgE­dot blotting and IgE­ELISA with synthetic polypeptides and HDM­allergic sera. Three­dimensional (3D) structural modeling was used to analyze the position of the immunodominant IgE epitope. The amino acid sequence homology between Der f 24 and the human UQCRB protein was determined to be 39.34%. IgE­ELISA and western blot analysis showed that all of the Der f­human UQCRB hybrid proteins generated, except for the one lacking 59 residues of the N­terminal region of Der f 24, were bound by allergic serum IgE. A synthetic polypeptide consisting of 32 residues of the N­terminal reacted with IgEs from HDM­allergic sera and could be used to generate high titer specific IgG or specific IgE antibodies in immunized mice. The 32­aa N­terminal region of Der f 24 was localized to a structural protrusion, which may facilitate specific IgE­binding. These results indicate that the immunodominant IgE epitope of Der f 24 is located mainly in a 32­residue region of the N­terminus. These findings may inform the mechanisms of HDM allergy sensitization and allergy immunotherapy development.

Identification of immunodominant IgE binding epitopes of Der p 24, a major allergen of Dermatophagoides pteronyssinus.

BACKGROUND: The identification of house dust mite (HDM) allergens and epitopes is important for allergy diagnosis and treatment. We sought to identify the Dermatophagoides pteronyssinus group 24 allergen (Der p 24) and to identify its immunodominant IgE epitope(s). METHODS: Der p 24 cDNA was cloned and expressed in a pET expression system. The IgE binding activity of purified recombinant (r)Der p 24 was evaluated by western blotting. Truncated Der p 24 proteins and overlapping synthetic polypeptides were subjected to IgE binding assays. Balb/c mice were immunized to investigate IgE epitope induction of IgE production. IgE binding of the 32 N-terminal residues of Der p 24 was compared to other Der p epitopes in enzyme-linked immunosorbent assays and dot blot assays. Human skin prick tests (SPTs) were performed. RESULTS: We cloned and expressed Der p 24 cDNA (GenBank accession no. KP893174.1). HDM allergic sera bound rDer p 24 in vitro and 5/10 HDM allergic patients (50%) had positive SPT reactions to rDer p 24. The immunodominant IgE epitope of Der p 24 was localized to the N-terminal 32-residue region, which produced a high specific IgE antibody titer in vivo and promoted mast cell ß-hexosaminidase release. The IgE binding activity this N-terminal epitope of Der p 24 was stronger than that of Der p 1 or Der p 2 IgE epitopes. CONCLUSIONS: We identified Der p 24 as a major HDM allergen with strong IgE binding activity via an immunodominant IgE epitope in the N-terminal 32-residue region, which triggers IgE production in vivo. The identified Der p 24 epitope may support HDM allergy diagnosis and treatment.