Polyamine Transport Protein PotD Protects Mice against Haemophilus parasuis and Elevates the Secretion of Pro-Inflammatory Cytokines of Macrophage via JNK–MAPK and NF–κB Signal Pathways through TLR4
Abstract
:1. Introduction
2. Materials and Methods
2.1. Animals and Ethics Statement
2.2. Strains, Plasmids, Primers and Bacterial Growth Conditions
2.3. Expression/Purification of Recombinant PotD Protein, and Removal of Endotoxin Contamination
2.4. Immunization
2.5. Analysis of Cellular Immune Response of Mice Using Immunofluorescent (IF) Assay and Flow Cytometry
2.6. Analysis of Lymphocyte Proliferation and Detection of Cytokines Using ELISA
2.7. Mouse Challenge
2.8. Bacterial Clearance
2.9. Cell Line and Culture Conditions
2.10. Detected of Pro-Inflammatory Cytokines Induced by PotD Using ELISA and Quantitative Real-Time PCR
2.11. Toll-Like Receptors 2 (TLR2) and 4 (TLR4) Blocking Assay
2.12. Western Blotting Assay for Induction of Pro-Inflammatory Cytokines by PotD
2.13. Determination of Working Concentrations of Inhibitors Using MTT Assay
2.14. Effect of Inhibitor on the MAPK and NF-κB Signal Pathways
2.15. Statistical Analysis
3. Results
3.1. Purification of rPotD Protein, Endotoxin Elimination, and Polyclonal Antibody Preparation
3.2. Recombinant PotD Protein Mediates a Cellular Immune Response
3.3. Lymphocyte Proliferation Assay and Detection of Cytokines
3.4. rPotD-Vaccinated Mice Show Less Severe Pathological Signs in Multiple Tissues
3.5. rPotD+Adjuvant-Vaccinated Mice Showed Significantly Elevated Bacterial Clearance Ability
3.6. rPotD Actively Elevates the Levels of Pro-Inflammatory Cytokines IL-1β, IL-6, and TNF-α
3.7. TLR4, Rather Than TLR2, Participates in Regulatory Role on Expression of Pro-Inflammatory Cytokines in Macrophages
3.8. Determination of Working Concentrations of Inhibitors Using MTT Assay
3.9. Production of Pro-Inflammatory Cytokines Mainly through JNK–MAPK and NF–κB Signal Pathways
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Primers | Primer Sequences (5′→3′) | Products (bp) |
---|---|---|
rPotD expression primers | ||
P1 (potD-pET-F) | cagcaaatgggtcgcggatccAATGACACTGTACATCTTTATACTT | 1015 |
P2 (potD-pET-R) | ctcgagtgcggccgcaagcttGCTTCGCAGCTTTTAACTCTTG | |
qPCR primers | ||
P3(GAPDH-F) | GTGTTCCTACCCCCAATGTG | 189 |
P4(GAPDH-R) | CATCGAAGGTGGAAGAGTGG | |
P5(IL-1β-F) | GGGCCTCAAAGGAAAGAATC | 183 |
P6(IL-1β-R) | TACCAGTTGGGGAACTCTGC | |
P7(IL-6-F) | GGGACTGATGCTGGTGACAA | 147 |
P8(IL-6-R) | TCCACGATTTCCCAGAGAACA | |
P9(TNF-α-F) | CGTCAGCCGATTTGCTATCT | 184 |
P10(TNF-α-F) | CTTGGGCAGATTGACCTCAG |
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Dai, K.; Ma, X.; Yang, Z.; Chang, Y.-F.; Cao, S.; Zhao, Q.; Huang, X.; Wu, R.; Huang, Y.; Yan, Q.; et al. Polyamine Transport Protein PotD Protects Mice against Haemophilus parasuis and Elevates the Secretion of Pro-Inflammatory Cytokines of Macrophage via JNK–MAPK and NF–κB Signal Pathways through TLR4. Vaccines 2019, 7, 216. https://doi.org/10.3390/vaccines7040216
Dai K, Ma X, Yang Z, Chang Y-F, Cao S, Zhao Q, Huang X, Wu R, Huang Y, Yan Q, et al. Polyamine Transport Protein PotD Protects Mice against Haemophilus parasuis and Elevates the Secretion of Pro-Inflammatory Cytokines of Macrophage via JNK–MAPK and NF–κB Signal Pathways through TLR4. Vaccines. 2019; 7(4):216. https://doi.org/10.3390/vaccines7040216
Chicago/Turabian StyleDai, Ke, Xiaoyu Ma, Zhen Yang, Yung-Fu Chang, Sanjie Cao, Qin Zhao, Xiaobo Huang, Rui Wu, Yong Huang, Qigui Yan, and et al. 2019. "Polyamine Transport Protein PotD Protects Mice against Haemophilus parasuis and Elevates the Secretion of Pro-Inflammatory Cytokines of Macrophage via JNK–MAPK and NF–κB Signal Pathways through TLR4" Vaccines 7, no. 4: 216. https://doi.org/10.3390/vaccines7040216