The Negative Regulation Mechanism Of Dermatophagoides Farina Protein Disulfide Isomerase In Allergic Asthma

Background: With the acceleration of urbanization and the rise of global temperature,the widespread use of air conditioners provides appropriate conditions for the growth and reproduction of HDM.At the same time,due to the acceleration of urbanization,people’s indoor activities are prolonged,resulting in a significant increase in HDM sensitization rate.The incidence of allergic diseases such as allergic rhinitis,allergic asthma,allergic conjunctivitis,allergic rash,allergic urticaria,and allergic conjunctivitis caused by dust mites increased accordingly.In the case of allergic asthma,about 80% of asthma patients are allergic to dust mites.At present,most of the researches on dust mite allergies focus on discovering allergens and exploring their sensitization mechanisms.In addition to the allergens that have been discovered,HDM also contains a large number of non-allergens.During the previous study of our group,we found that the Pplase protein can synergistically induce allergic asthma.As a newly discovered non-allergen,whether PDI cooperates with allergens in allergic asthma and the regulatory mechanism of PDI in allergic asthma is unknown.Objective: To clone and express PDI protein of Dermatophagoides farinae,identify its immunogenicity and study its mechanism in allergic asthma.Methods:⑴ The PDI gene was synthesized by genetic engineering technology,and then we constructed the recombinant plasmid pET32a(+)-PDI-Origami.The recombinant protein rPDI was purified by Ni2+ affinity chromatography.⑵ The immunogenicity of recombinant protein r-PDI was detected by immunoblotting(WB),indirect enzyme-linked immunosorbent assay(ELISA),and clinical skin prick test(SPT).⑶ An allergic asthma model of PDI and allergen synergistic sensitization was constructed using Der f1 or OVA as the main allergens to study the role of PDI in asthma.⑷ Cellular and animal experiments were conducted to investigate the mechanism of PDI proteins when they act synergistically with allergens.Results: ⑴ Cloning,expression and purification of recombinant PDI protein,SDSPAGE showed that the molecular weight of r-PDI protein was 74 KDa.⑵ The clinical prick test(SPT)showed no PDI-positive reaction in 30 dust mite allergic patients;ELISA showed that among 35 dust mite allergic patients,4 patients showed PDI-specific IgE positive,and the positive rate was 12%;WB test showed that among the 9 patients with dust mite allergies,1 patient was positive for PDI-specific IgE,and the positive rate was 11%;the positive rate did not meet the requirements for becoming an allergen,and it was regarded as a dust mite nonallergen.⑶ When PDI works synergistically with the allergen Der f1 or OVA,it can reduce allergen-induced serum-specific IgE,decrease the expression of Th2-type inflammatory cytokines,decrease the number of eosinophils,and reduce the pathological changes in the lungs,and then negatively regulate allergic asthma.⑷ In vitro experiments showed that PDI can induce DC to differentiate into CD103+DCreg,with a high expression of costimulatory molecule CD80 and a secretion of a large amount of IL10,which leads to high expression of CTLA-4 on T cell surface,that competitively inhibit the activation of CD28,and induce T cell to polarize into Treg,cause negative regulation of inflammatory responses by allergens.Conclusion: PDI is a NON-ALLERGEN of Dermatophagoides farinae.When it cooperates with allergens,it can negatively regulate the inflammatory reaction of allergic asthma caused by allergens.