ABSTRACT
Objective To evaluate the effects of recombinant fusion protein interleukin-17F/His (IL-17F/His) on expression of immune-related inflammatory factors in murine models with Streptococcus pneumoniae (SP) infection.Methods BALB/c mice were randomly divided into SP infection group,fusion protein intervention group and the normal control group.Before intranasal infection with SP,the mice were treated with PBS or IL-17F/His respectively.The number of bacteria and leukocytes in bronchoalveolar lavage fluid (BALF) was counted,and the mRNA levels of β-defensin-2 (Defb2),macrophage inflammatory protein (MIP)-lα and MIP-2β in lung tissue were detected by real-time fluorescent quantitative PCR,the concentrations of MIP-1 α,MIP-2β,IFN-γ and IL-4 in BALF,supematants of spleen cell and mediastinal lymph node cell were assayed by enzyme linked immunosorbent assay.The expressions of IL-17F and Defb2 in lung tissues were observed with immunocytochemistry.Results 1.Compared with the SP infection group,in the fusion protein intervention group,the total number of WBC (209.00 ± 18.23) was higher,the number of neutrophil,macrophage and lymphocyte[(8.67 ±2.16) × 106/L,(193.50 ± 16.50) × 106/L,(6.83 ± 1.17) × 106/L] were higher in BALF(U/F =38.097,28.854,32.942,27.810,all P ＜ 0.05),while the number of bacteria [2.75 (1.15-3.09) × 103/L] was significantly lower(P =0.02).2.Compared with SP infection group,the expression levels of Defb2 mRNA and MIP-1α mRNA(31.64 ±4.97,5.81 ± 1.09) in lung tissues were higher in the fusion protein intervention group,while the expression level of MIP-2β mRNA (6.69 ± 2.05) was lower (t =4.889,2.306,3.536,all P ＜ 0.05).3.Compared with the SP infection group,the concentration of MIP-2β [(64.15 ± 13.41) ng/L] was significantly decreased and concentration of IL-4 [(92.28 ± 4.52) ng/L] was significantly increased in BALF in the fusion protein intervention group(H/F =159,289,40.767,all P ＜0.01).4.Compared with the SP infection group,the concentrations of MIP-2β and IL-4 [(255.02 ± 13.95) ng/L,(107.02 ± 7.53) ng/L] were both significantly increased in spleen cell culture supematants in the fusion protein intervention group,the concentrations of MIP-1 α and IFN-γ [(413.61 ± 24.23) ng/L,(98.88 ± 5.84) ng/L] were both significantly decreased (all P ＜ 0.05).5.Immunocytochemistry analysis revealed that the expressions of IL-17F and Defb2 were both higher in the fusion protein intervention group than those in the SP infection group; there was no expression in the normal control group.Conclusions Intranasal inoculation of recombinant fusion protein IL-17F/His can promote pulmonary neutrophil and macrophage recruitment and increase bacterium clearance,and enhance the host defense against SP infection through increa-sing the expression of defensins,regulating the levels of MIP,IL-4 and IFN-γ.
ABSTRACT
ObjectiveTo evaluate the effects of intranasal administration of recombinant interleukin-17A(rIL-17A) on the expressions of β-Defensin-2(Defb2) and macrophage inflammatory protein(MIP) in pneumococcal pneumonia murine models.MethodsTwenty-four BALB/c mice were divided randomly into normal control,pneumococcal pneumonia,and rIL-17A intervention groups ( n =8 ).Before intranasal (i.n) infection with Streptococcus pneumoniae,the mouse was treated with PBS or rIL-17Ai.n respectively.The mRNA levels of Defb2,MIP-1α and MIP-2β expression in lung tissue were detected by real-time quantity PCR.The numbers of bacteria and leukocytes in bronchoalveolar lavage fluid (BALF) were counted as well.And the concentrations of MIP-1α,MIP-2β,IFN-γ and IL-4 in BALF and in supematants of spleen cells and mediastinal lymph node cells were assayed by ELISA.Changes in lung tissue histopathology were observed with HE staining through light microscope.ResultsNeutrophil and macrophage numbers are higher in BALF of rIL-17A group,while the numbers of bacteria were lower,when compared with those in pneumonia group( P＜0.01 ).The expression of Defb2 and MIP-1α mRNA were up-regulated in lung after rIL17A treatment(P＜0.01 ).When compared with rIL-17A non-treated mice,rIL-17A treated mice secretedhigher levels of MIP-1α in lymph node cell culture supernatants( P＜0.01 ),higher levels of MIP-2β were observed in spleen cell and lymph node cell culture supernatants( P＜0.01 ),higher levels of IFN-T were detected in BALF( P ＜ 0.01 ) and culture supernatants of spleen cell ( P ＜ 0.01 ) and lymph node cell ( P ＜0.05),and higher levels of IL-4 were detected in BALF and spleen cell culture supernatant(P＜0.01 ).Comparative analysis have not detect a significant irflammatory cell increases in rIL-17A treated mice lung tissue; however the histopathological lesions were decreased.ConclusionIntranasal inoculation of rIL-17A can promote pulmonary neutrophil and macrophage recruitment and bacterium clearance,Intranasal inoculation of riL-17enhances the host defense against Streptococcus pneumoniae infection partly through increasing the expression levels of defensins,MIP,IFN-T and IL-4 etc.
ABSTRACT
Objective To express and purify mouse interleukin 17A(mIL-17A) in E. coli and to analyze its ability of stimulating macrophage inflammatory factors expression. Methods The coding gene of mouse mIL-17A mature protein was amplified from mouse spleen cells by RT-PCR. PCR product was cloned into the prokaryotic expressing vector pET28a, and the resulting recombinant plasmid pET28a/mIL-17a was then transformed into the host E. coli strain BL21(DE3) for expression. The mIL-17A protein was identified by SDS-PAGE and Western blot. The recombinant protein was purified by the Ni-NTA affinity chromatography, and was further tested on the stimulation of cytokine and chemokine of RAW264.7 cells by ELISA and real-time quantity PCR in vitro. Results The mIL-17A with bioactivity was over-expressed and purified successfully, and the results of real-time PCR and ELISA showed that recombinant mIL-17A stimulated macrophage mRNA upregulation of IL-6, defensin β2 and Cxcl3 and secretion of defensin β2, Ccl3, Cxcl3,IFN-γ, IL-6 and IL-4. Interestingly, these effects could be blocked by the addition of anti-IL-17A neutralizing antibody partly. After treatment with mIL-17, 74. 87-fold of defensin β2 mRNA expression was increased comparing with that of untreated cells( P ＜0.01 ), while blocking with anti-IL-17A antibody the increase was only 5.4-fold(P ＜ 0.01 ). Conclusion The recombinant mIL-17A has a strong stimulation on secretion of cytokine and chemokine of macrophage, that maybe result to the enhancement of anti-infection ability of macrophage.