Notch Signaling Regulates the Function and Phenotype of Dendritic Cells in Helicobacter pylori Infection
Abstract
:1. Introduction
2. Materials and Methods
2.1. H. pylori Culture
2.2. Generation of Murine Bone Marrow-Derived DCs
2.3. Stimulation of BMDCs with H. pylori
2.4. Co-Culture of BMDCs and CD4+ T Cells
2.5. RNA Extraction and Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
2.6. Protein Extraction and Western Blot
2.7. ELISA
2.8. Flow Cytometry
2.9. Statistical Analysis
3. Results
3.1. H. pylori Induced the Expression of All Notch Receptors and Notch Ligands Dll4 and Jagged1 in BMDCs
3.2. Notch Signaling Was Involved in the Regulation of Cytokines in H. pylori-Infected BMDCs
3.3. Notch Signaling Was Involved in Regulating the Phenotype of BMDCs during H. pylori Infection
3.4. Inhibition of Notch Signaling in BMDCs Shifted the Th17/Treg Balance toward Treg of CD4+ T Cells
4. Discussions
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
References
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Species | Target Gene | Primer Sequence (5′→3′) | |
---|---|---|---|
Murine | βActin | Forward | GCAGGAGTACGATGAGTCCG |
Reverse | ACGCAGCTCAGTAACAGTCC | ||
Murine | Notch1 | Forward | ACGTAGTCCCACCTGCCTAT |
Reverse | CAGGTGCCCTGATTGTAGCA | ||
Murine | Notch2 | Forward | GTTGATCCCCGTCAGTGTGT |
Reverse | CAGGAGGCTGAAGTCGGTTT | ||
Murine | Notch3 | Forward | ACTCCTCCTCAGGGAGATGC |
Reverse | GTGGGGTGAAGCCATCAGG | ||
Murine | Notch4 | Forward | CCAGAGAGCTTCTGTGTGGA |
Reverse | CAGAAATCCAGGGGCACACT | ||
Murine | Dll1 | Forward | ACCAAGTGCCAGTCACAGAG |
Reverse | TCCATCTTACACCTCAGTCGC | ||
Murine | Dll3 | Forward | CTCCCGGATGCACTCAACAA |
Reverse | TGGAAGGGGCTGGTATGACA | ||
Murine | Dll4 | Forward | CTTTGGCAATGTCTCCACGC |
Reverse | ACTGCCGCTATTCTTGTCCC | ||
Murine | Jagged1 | Forward | GGGTCAGTTTGAGCTGGAGA |
Reverse | GTACGTATCACACTCGTCGC | ||
Murine | Jagged2 | Forward | GCCTCGTCGTCATTCCCTTT |
Reverse | AGCTCCTCATCTGGAGTGGT | ||
Murine | Il6 | Forward | TAGTCCTTCCTACCCCAATTTCC |
Reverse | TTGGTCCTTAGCCACTCCTTC | ||
Murine | Il12 | Forward | CAATCACGCTACCTCCTCTTTT |
Reverse | CAGCAGTGCAGGAATAATGTTTC | ||
Murine | Il1β | Forward | TTCAGGCAGGCAGTATCACTC |
Reverse | GAAGGTCCACGGGAAAGACAC | ||
Murine | Tnfα | Forward | GGTCACTGTCCCAGCATCTT |
Reverse | CTGTGAAGGGAATGGGTGTT | ||
Murine | Il10 | Forward | ATTTCCGATAAGGCTTGGCAA |
Reverse | GCTGGACAACATACTGCTAACC | ||
Murine | Tgfβ | Forward | AGTGTGGAGCAACATGTGGAACT |
Reverse | AGCAGCCGGTTACCAAGGTA | ||
Murine | Tbx21 | Forward | AACACACACGTCTTTACTTTCCA |
Reverse | CGTATCAACAGATGCGTACATGG | ||
Murine | Ifnγ | Forward | ACTGGCAAAAGGATGGTGAC |
Reverse | ACCTGTGGGTTGTTGACCTC | ||
Murine | Gata3 | Forward | CGAGATGGTACCGGGCACTA |
Reverse | GACAGTTCGCGCAGGATGT | ||
Murine | Il4 | Forward | TCTCGAATGTACCAGGAGCCATAT |
Reverse | AAGCACCTTGGAAGCCCTACAGA | ||
Murine | Rorγt | Forward | CCGCTGAGAGGGCTTCAC |
Reverse | TGCAGGAGTAGGCCACATTACA | ||
Murine | Il17A | Forward | TTTAACTCCCTTGGCGCAAAA |
Reverse | CTTTCCCTCCGCATTGACAC | ||
Murine | Il17F | Forward | TGCTACTGTTGATGTTGGGAC |
Reverse | AATGCCCTGGTTTTGGTTGAA | ||
Murine | Foxp3 | Forward | CCCATCCCCAGGAGTCTTG |
Reverse | ACCATGACTAGGGGCACTGTA |
Antibodies | Dilution or Concentration | Source (Location) |
---|---|---|
Rabbit monoclonal anti-Jagged1 | 1:1000 | Cell signaling, Danvers, MA, USA Cat. No. 2620S |
Rabbit polyclonal anti-Dll4 | 1:1000 | Affinity, San Francisco, CA, USA Cat. No. DF13221 |
Anti-Dll1 antibody | 1:1000 | Abcam, Cambridge, UK No. ab84620 |
β-Tubulin (9F3) Rabbit mAb | 1:1000 | Cell signaling, Danvers, MA, USA Cat. No. 2128S |
Peroxidase conjugated Goat anti-Rabbit IgG | 1:10,000 | Fude BioTech, Hangzhou, China Cat. No. FDR007 |
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Liu, Q.; Chen, C.; He, Y.; Mai, W.; Ruan, S.; Ning, Y.; Li, Y. Notch Signaling Regulates the Function and Phenotype of Dendritic Cells in Helicobacter pylori Infection. Microorganisms 2023, 11, 2818. https://doi.org/10.3390/microorganisms11112818
Liu Q, Chen C, He Y, Mai W, Ruan S, Ning Y, Li Y. Notch Signaling Regulates the Function and Phenotype of Dendritic Cells in Helicobacter pylori Infection. Microorganisms. 2023; 11(11):2818. https://doi.org/10.3390/microorganisms11112818
Chicago/Turabian StyleLiu, Qiaoyuan, Chuxi Chen, Yunxuan He, Wenhao Mai, Shipeng Ruan, Yunshan Ning, and Yan Li. 2023. "Notch Signaling Regulates the Function and Phenotype of Dendritic Cells in Helicobacter pylori Infection" Microorganisms 11, no. 11: 2818. https://doi.org/10.3390/microorganisms11112818