Mechanisms Of Regulation On Innate Immune Response In Respirotory Tract During Treatemt With Dust Mite Vaccine

Objective: Recently research found that bronchial asthma had suppressed the pulmonary innate immune system and could result in increased susceptibility to colonization and infection. Allergen specific immunotherapy(SIT) has effectively therapeutical effect on allergic asthma, but the relation between SIT and airway innate immune has been reported little. This study plans to illuminate the effects of standardized house dust mite vaccine on airway innate immunity, and to discuss the mechanism. Methods: 1. animal experiments:①Female BALB/c mice(4–6 wk. old) Mice were sensitized by three i.p. injections and challenged seven times intranasally. SIT group were immunotherapy by eight subcutaneous injections before their challenge. Control animals received PBS alone. Twenty-four hours after the last exposure to allergen, mice were euthanized, and AHR were analyzed; bronchoalveolar lavage(BAL) was performed to count cell numbers, and histopathologic examination were performed. HDM-specific antibody responses were evaluated using ELISA.②Female BALB/c mice(4–6 wk. old) Mice were sensitized by three i.p. injections and challenged seven times intranasally. SIT group were immunotherapy by eight subcutaneous injections before their challenge. Control animals received PBS alone. Twenty-four hours after the last exposure to allergen, mice were slightly anesthetized by diethyl ether and infected intranasally with P.aeruginosa in PBS or PBS alone as control. Twenty-four hours after the infection,rectal temperature was taken;bronchoalveolar lavage(BAL) was performed to count cell numbers and to observe bacteria loads. Histopathologic analysis were evaluated, and quantity of bacteria in lung homogenate were calculated.③Female BALB/c mice(4–6 wk. old) Mice were sensitized by three i.p. injections and challenged seven times intranasally. SIT group were immunotherapy by eight subcutaneous injections before their challenge. Control animals received PBS alone. Twenty-four hours after the last exposure to allergen, mice were euthanized, concentration of CRAMP, IL-4, IFN-γ, IL-10 and TGF-β1 in BAL F were determined by ELISA.IF or q PCR was used to evaluate the expression of CRAMP in lung. CYP27b1 expression was observed by IHC.2. cell experiments:①Human bronchial epithelial cells were incubated with 25(OH)2 VD3,TGF-β1+25(OH)2 VD3,IFN-γ+25(OH)2 VD3,IL-10+25(OH)2 VD3,TGF-β1,IFN-γ,IL-10 and 1,25(OH)2 VD3 respectively or control medium. CAMP expression was determined using q PCR.②CYP27b1 expression of HBE cells were observed by q PCR and western blot after incubating with different levels of TGF-β1 or control medium.③After preincubation with ITRA, 25(OH)2 VD3 and TGF-β1+25(OH)2 VD3 were added to HBE cells, then the expression of CAMP were evaluated using q PCR and western blot. ④After preincubation with ITRA, 25(OH)2 VD3 and TGF-β1+25(OH)2 VD3 were added to HBE cells, then them were infected with P.aeruginosa. The cells were homogenized and spread on LB plates to determine levels of intracellular bacteria.Resuts:1. animal experiments:①Mice treated with SIT showed a significant decrease in AHR when compared with the non-treated group(P<0.01); cell amounts in BAL F reduced significantly; SIT induced increase of Ig G2a(P<0.01) and decreased s Ig E and Ig G1 levels(both P<0.01) in accordance with alleviating inflammation of lungs.②Temperature was lower in SIT mice(P<0.01), and cell numbers and bacteria loads in BAL F reduced significantly(both P<0.01). Inflammation of lungs were alleviated, and bacteria amounts in airway also decreased(P<0.01).③CRAMP expression were upgraded by SIT in both BAL F and airway epithelium(all P<0.01). IFN-γ, TGF-β1 and IL-10 levels were upgraded(all P<0.01) while IL-4 were reduced(P<0.01). Expression of CYP27b1 was increased in airway of SIT-treated mice. 2. cell experiments:①TGF-β1+25(OH)2 VD3, 25(OH)2 VD3 and 1,25(OH)2 VD3 upregulated m RNA of CAMP on HBE cells(all P<0.01),and TGF-β1+25(OH)2 VD3 was more efficient than 25(OH)2 VD3(P<0.05).②Two doses, 1ng/ml and 10ng/ml,increased CYP27b1 expression on HBE cells(all P<0.01).③ITRA reduces the upgrading effects of TGF-β1+25(OH)2 VD3 on CAMP expression(all P<0.01).④TGF-β1+25(OH)2 VD3(P<0.01)and 25(OH)2 VD3(P<0.05)cut down the intracellular bacteria loads, and efficacy of TGF-β1+25(OH)2 VD3 was higher than 25(OH)2 VD3(P<0.05), while ITRA inhibited the up-regulation(P<0.05).Conclusion: Immunotherapy with HDM improved pulmonary host defense to bacteria by up-regulating cathelicidin related antimicrobial peptide in allergic inflammation mice, and the mechanism could conclude the increased expression of CYP27b1 by TGF-β1 stimulating.