RESUMO
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.
Assuntos
Aspergillus fumigatus/enzimologia , Endorribonucleases/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Células Th2/citologia , Células Th2/metabolismo , Animais , Endorribonucleases/genética , Feminino , Interleucina-10/metabolismo , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Células RAW 264.7 , Células Th2/imunologiaRESUMO
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.
Assuntos
Alérgenos/imunologia , Antígenos de Dermatophagoides/imunologia , Dermatophagoides farinae/imunologia , Epitopos Imunodominantes/imunologia , Imunoglobulina E/imunologia , Pyroglyphidae/imunologia , Adolescente , Adulto , Animais , Proteínas de Artrópodes/imunologia , Linhagem Celular Tumoral , Feminino , Humanos , Hipersensibilidade/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Ratos , Adulto JovemRESUMO
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.