Construction Of Recombinant Mycobacterium Smegmatis Expressing Ag85A-IL-17A And Its Mechanisms Against Airway Inflammation

BACKGROUND: Airway inflammation is a common pathological feature of a variety of respiratory diseases, including infectious airway inflammation and allergic airway inflammation. Streptococcus pneumoniae is a common respiratory pathogen, resulting in neutrophilic airway inflammation. Asthma is a chronic inflammatory disease characterized by airway inflammation, airway hyperresponsiveness and mucus hyperproduction, and neutrophils play an important role in the pathological process of asthma. Accumulating evidence indicates that IL-17 A and IL-17 F contribute to the development of neutrophilic inflammation. Therefore, inhibiting or antagonizing the function of IL-17 A and IL-17 F has become an important method against airway inflammation. We found that IL-17 A and IL-17 F could enhance infectous inflammation caused by Streptococcus pneumoniae, so we intend to further study on the relationship between IL-17 A and IL-17 F and allergic airway inflammation. According to the theory of Lanre Delavallée, in order to induce a B cell response and to obtain autoantibodies to neutralize self-cytokines, cytokines should be modified with foreign Th cell epitopes. Hence, we speculate it would be beneficial for asthma treatment to induce the autoantibody to IL-17 A and IL-17 F in vivo, which could suppress or block the functions of IL-17 A and IL-17 F. Mycobacterium smegmatis(M. smegmatis) is a non-pathogenic saprophyte with immunoregulatory function, and it acts as an effective vaccine vector. Ag85 A is one of three proteins that together make up the Ag85 complex, and it could stimulate Th1-type cellular immunity. In this study, Mycobacterium smegmatis was used as the vector and Ag85 A provided Th cell epitopes for inducing IL-17 A and IL-17 F autoantibody, and the mechanisms of autoantibody against airway inflammation were further investigated.OBJECTIVE: The aim of this research was to construct recombinant Mycobacterium smegmatis expressing Ag85A-IL-17A/F(r MS-Ag85a-IL-17a/f) and induce autoantibody of IL-17A/F in vivo, and further investigate the mechanisms of r MS-Ag85a-IL-17a/f against airway inflammatory response in mice.METHODS: There were three stages of this experiment:(1) Construction of r MS-Ag85a-IL-17a/f: Recombinant shuttle plasmid p MFA42S-Ag85a-IL-17a/f was constructed by inserting fusion gene Ag85a-IL-17a/f into shuttle vector p MFA42 S, which was transformed to M. smegmatis by electroporation to obtain recombinant M. smegmatis named r MS-Ag85a-IL-17a/f. Research on autoantibody induced by r MS-Ag85a-IL-17a/f in vivo: Mice were intranasally immunized with M. smegmatis or recombinant M. smegmatis, and antibody response in sera and the concentrations of cytokines in lung homogenates were detected by enzyme-linked immunosorbent assay(ELISA). Nitric oxide secretion was determined using Griess reagent. The m RNA expressions of transcription factor in lung tissue were detected by Realtime PCR. Lung tissue sections were stained with hematoxylin and eosin(HE) staining to assess the infiltration of inflammatory cells.(2) Analysis of the functions of r MS-Ag85a-IL-17 a on macrophage in vitro: Macrophage was infected with r MS-Ag85a-IL-17 a in vitro, and the effects of recombinant M. smegmatis on the phagocytic function and secretion function were analyzed.(3) Investigating the mechanisms of r MS-Ag85a-IL-17 a against asthmatic airway inflammation: Mice were randomly divided into five groups of six animals: PBS group, asthma group, severe asthma group, severe asthma plus M. smegmatis group and severe asthma plus r MS-Ag85a-IL-17 a group. The number of leukocyte in bronchoalveolar lavage fluid was counted by hemacytometer and differential cell counts were performed using Wright-Giemsa stain. Lung tissue sections were stained with hematoxylin and eosin and periodic acid-Schiff(PAS) staining to assess the infiltration of inflammatory cells and the secretion of mucus. ELISA was performed to detect the concentrations of cytokines in BALF and cell culture supernatants of spleen. The percentage of INF-γ+ Th1 cells and CD4+ Th2 cells in spleen was determined by flow cytometry. The m RNA expressions of inflammatory mediators in lung tissue were analyzed by Realtime PCR.RESULTS:(1) Recombinant shuttle plasmid p MFA42S-Ag85a-IL-17 a was successfully constructed and transformed to M. smegmatis by electroporation. The expression of fusion protein Ag85A-IL-17 A was verified by Western blot, which was specifically bound to IL-17 A antibody. A high titer of antibody specific to IL-17 A was detected in sera from mice immunized with r MS-Ag85a-IL-17 a, and the antibody emerged at day 7 and peaked the highest titer at day 21, which lasted for more than 4 weeks. r MS-Ag85a-IL-17 a significantly decreased the concentrations of IL-5, IL-17 A and increased the concentration of IL-12 in lung homogenates; meanwhile up-regulated the expression of T-bet in lung tissue. r MS-Ag85a-IL-17 f could not induce effective autoantibody of IL-17 F. Recombinant IL-17 F enhanced the inflammatory response and reduced Streptococcus pneumoniae colonization in lung.(2) r MS-Ag85a-IL-17 a promoted macrophage phagocytosis and apoptosis, and significantly increased IL-6, macrophage inflammatory protein(MIP)-1α and tumor necrosis factor(TNF)-α levels as well as up-regulated IL-6, IL-10, β-defensin-2(Defb-2) and inducible nitric oxide synthase(i NOS) m RNA expressions in lung tissue.(3) Compared with severe asthma group, r MS-Ag85a-IL-17 a immunization significantly decreased the numbers of total leukocytes, neutrophils, eosinophils and the concentrations of IL-5, IL-6, IL-13, IL-17 A in BALF and cell culture supernatants of spleen; increased the concentrations of IL-12 in BALF and IL-12, IL-10 in cell culture supernatants of spleen; inhibited the myeloperoxidase(MPO) vitality in BALF; down-regulated the expressions of IL-6, IL-23, Eotaxin-1, MIP-2, TGF-β, GATA3 and up-regulated the expressions of IL-10 and T-bet; the results of flow cytometry showed that r MS-Ag85a-IL-17 a immunization promoted the INF-γ+ Th1 cells and suppressed CD4+ Th2 cells in spleen; the histopathological findings showed that r MS-Ag85a-IL-17 a immunization significantly reduced the infiltration of inflammatory cells and the secretion of mucus in airway of asthmatic mice.CONCLUSIONS:(1) r MS-Ag85a-IL-17 a was successfully constructed and the expressed fusion protein Ag85A-IL-17 A could specifically bind to IL-17 A antibody; r MS-Ag85a-IL-17 a could induce autoantibody of IL-17 A in vivo;(2) r MS-Ag85a-IL-17 a could promote macrophage phagocytosis and apoptosis and enhance the function of macrophage against infection;(3) r MS-Ag85a-IL-17 a immunization could attenuate airway inflammation in mice.