TLR2 and TLR4 signaling pathways are required for recombinant Brucella abortus BCSP31-induced cytokine production, functional upregulation of mouse macrophages, and the Th1 immune response in vivo and in vitro.
Brucella abortus is a zoonotic Gram-negative pathogen that causes brucelosis in ruminants and humans. Toll-like receptors (TLRs) recognize Brucella abortus and initiate antigen-presenting cell activities that affect both innate and adaptive immunity. In this study, we focused on recombinant Brucella cell-surface protein 31 (rBCSP31) to determine its effects on mouse macrophages. Our results demonstrated that rBCSP31 induced TNF-α, IL-6 and IL-12p40 production, which depended on the activation of mitogen-activated protein kinases (MAPKs) by stimulating the rapid phosphorylation of p38 and JNK and the activation of transcription factor NF-κB in macrophages. In addition, continuous exposure (>24 h) of RAW264.7 cells to rBCSP31 significantly enhanced IFN-γ-induced expression of MHC-II and the ability to present rBCSP31 peptide to CD4(+) T cells. Furthermore, we found that rBCSP31 could interact with both TLR2 and TLR4. The rBCSP31-induced cytokine production by macrophages from TLR2(-/-) and TLR4(-/-) mice was lower than that from C57BL/6 macrophages, and the activation of NF-κB and MAPKs was attenuated in macrophages from TLR2(-/-) and TLR4(-/-) mice. In addition, CD4(+) T cells from C57BL/6 mice immunized with rBCSP31 produced higher levels of IFN-γ and IL-2 compared with CD4(+) T cells from TLR2(-/-) and TLR4(-/-) mice. Macrophages from immunized C57BL/6 mice produced higher levels of IL-12p40 than those from TLR2(-/-) and TLR4(-/-) mice. Furthermore, immunization with rBCSP31 provided better protection in C57BL/6 mice than in TLR2(-/-) and TLR4(-/-) mice after B. abortus 2308 challenge. These results indicate that rBCSP31 is a TLR2 and TLR4 agonist that induces cytokine production, upregulates macrophage function and induces the Th1 immune response.
State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing, China.
Jia-Yun
JY
Li
State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing, China.
Yuan
Y
Liu
State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing, China.
Xiao-Xue
XX
Gao
Model Animal Research Center, Nanjing University, Nanjing, China.
Xiang
X
Gao
State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing, China.
Hong
H
Cai
D000818:Animals; D017951:Antigen Presentation / Q000235:genetics; D000942:Antigens, Bacterial / Q000276:immunology*; D001426:Bacterial Proteins / Q000276:immunology*; D002003:Brucella abortus / Q000276:immunology*; D002006:Brucellosis / Q000276:immunology*; D016207:Cytokines / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D018836:Inflammation Mediators / Q000378:metabolism; D008213:Lymphocyte Activation / Q000235:genetics; D020935:MAP Kinase Signaling System / Q000235:genetics; D008264:Macrophages / Q000276:immunology* / Q000382:microbiology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D016328:NF-kappa B / Q000378:metabolism; D011994:Recombinant Proteins / Q000276:immunology; D018417:Th1 Cells / Q000276:immunology* / Q000382:microbiology; D051195:Toll-Like Receptor 2 / Q000235:genetics / Q000378:metabolism*; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism*
C527376:31-kDa cell surface protein, Brucella abortus; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D016207:Cytokines; D018836:Inflammation Mediators; D016328:NF-kappa B; D011994:Recombinant Proteins; C493486:Tlr2 protein, mouse; C493487:Tlr4 protein, mouse; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4