Modulation of PTPN2/22 Function by Spermidine in CRISPR-Cas9-Edited T-Cells Associated with Crohn’s Disease and Rheumatoid Arthritis
Abstract
:1. Introduction
2. Materials and Methods
2.1. CRISPR-Cas9 Transfection, Stimulation, and Treatment of Jurkat T-Cells
2.2. Confirming Induction of PTPN2/22 SNPs
2.3. Measurement of PTPN2/22 Expression in Treated Jurkat T-Cells
2.4. T-Cell Proliferation Assay of Treated Jurkat T-Cells
2.5. Measurement of IFN-γ and TNF-α Secretion in Treated Jurkat T-Cells
2.6. CD25 (IL-2RA) Fluorescent Immunostaining Assay
2.7. Statistical Analysis
3. Results
3.1. PTPN2/22 SNP Sequencing
3.2. PTPN2/22 SNPs Decrease Gene Expression upon Induction in Jurkat T-Cells Using CRISPR-Cas9
3.3. PTPN2/22 SNPs Increase T-Cell Proliferation upon PHA Induction in Jurkat T-Cells Using CRISPR-Cas9
3.4. PTPN2/22 SNPs Increase Pro-Inflammatory Cytokine Secretion upon Induction in Jurkat T-Cells Using CRISPR-Cas9
3.5. PTPN2/22 SNPs Increase T-Cell Activation upon Induction in Jurkat T-Cells Using CRISPR-Cas9
3.6. Spermidine Increased PTPN2/22 Expression in CRISPR-Cas9-Edited Jurkat T-Cells with PTPN2/22 SNPs
3.7. Spermidine Decreases T-Cell Proliferation in CRISPR-Cas9-Edited Jurkat T-Cells with PTPN2/22 SNPs
3.8. Spermidine Decreases Pro-Inflammatory Cytokine Secretion in CRISPR-Cas9-Edited Jurkat T-Cells with PTPN2/22 SNPs
4. Spermidine Decreases T-Cell Activation in CRISPR-Cas9-Edited Jurkat T-Cells with PTPN2/22 SNPs
5. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Gene | RefSNP | Mutation | Location | Mean Allele Frequency | Mutation Phenotype | Reference |
---|---|---|---|---|---|---|
PTPN2 | rs478582 | T → C | Intron 3 | C = 0.3158 | High susceptibility to RA, TID, MS, and Celiac disease | [16] |
PTPN22 | rs2476601 | G → A | R620W | A = 0.0588 | High susceptibility to CD, RA, TID, MS, SLE, and Celiac disease | [20] |
Gene | Component | Sequence (5′ → 3′) |
---|---|---|
PTPN2 | sgRNA | AUUAUACUACGUCAAUUCAC +scaffold |
Donor DNA | CTACCTCAAGTAAAAAATGCATTTTAGTTTCCTGTGAATT GACGCAGTATAATAGCTCATAGTTACATTAATCTGCATAT (80 bases) | |
Donor DNA reverse complement | ATATGCAGATTAATGTAACTATGAGCTATTATACTGCGTC AATTCACAGGAAACTAAAATGCATTTTTTACTTGAGGTAG (80 bases) | |
Forward PCR Primer | CAGGGCTGTCTTTCCCCCTA (20 bases) | |
Reverse PCR Primer | GCAAAGTGTCCACCTTTGAT (20 bases) | |
PTPN22 | sgRNA | AAUGAUUCAGGUGUCCGUAC +scaffold |
Donor DNA | AGCTTCCTCAACCACAATAAATGATTCAGGTGTCC TACAGGAAGTGGAGGGGGGATTTCATCATCTATCC (70 bases) | |
Donor DNA reverse complement | GGATAGATGATGAAATCCCCCCTCCACTTCCTGTA GGACACCTGAATCATTTATTGTGGTTGAGGAAGCT (70 bases) | |
Forward PCR Primer | CGCCCAGCCCTACTTTTGAG (20 bases) | |
Reverse PCR Primer | CCATGCCCATCCCACACTTT (20 bases) |
Gene | Forward Primer Sequence (5′ → 3′) | Reverse Primer Sequence (5′ → 3′) |
---|---|---|
GAPDH | 5′-CTTTTGCAGACCACAGTCCATG-3′ (22 bases) | 5′-TTTTCTAGACGGCAGGTCAGG-3′ (21 bases) |
PTPN2 | 5′-CTAGAGGGTTAGCGAGCCTCA-3′ (21 bases) | 5′- TCATGTGGGAATGATTTTTGGTCAC-3′ (25 bases) |
PTPN22 | 5′-TAGTTTTATTTGCAGGTGTACTTGCAG-3′ (27 bases) | 5′-TGGTCAAGATGCTGCCTAACATT-3′ (23 bases) |
Cell Group | [IFN-γ] ± SD (pg/mL) | [TNF-α] ± SD (pg/mL) |
---|---|---|
WT | 0.029 ± 0.003 | 0.293 ± 0.042 |
PTPN2 (TT → CC) | 0.060 ± 0.004 * | 0.507 ± 0.014 * |
PTPN22 (GG → AA) | 0.054 ± 0.002 * | 0.408 ± 0.039 * |
Treatment | Spermidine Concentration (μM) | IFN-γ ± SD (pg/mL) | TNF-α ± SD (pg/mL) |
---|---|---|---|
WT | - | 0.029 ± 0.003 | 0.293 ± 0.042 |
WT+PHA | - | 0.283 ± 0.026 | 0.785 ± 0.012 |
10 | 0.124 ± 0.005 * | 0.359 ± 0.012 * | |
20 | 0.172 ± 0.010 * | 0.353 ± 0.022 * | |
PTPN2 | - | 0.060 ± 0.004 | 0.408 ± 0.039 |
PTPN2+PHA | - | 0.359 ± 0.020 | 4.87 ± 0.292 |
10 | 0.115 ± 0.010 * | 0.469 ± 0.015 * | |
20 | 0.164 ± 0.006 * | 0.484 ± 0.016 * | |
PTPN22 | - | 0.054 ± 0.002 | 0.507 ± 0.014 |
PTPN22+PHA | - | 0.237 ± 0.009 | 10.8 ± 0.318 |
10 | 0.108 ± 0.010 * | 0.609 ± 0.021 * | |
20 | 0.130 ± 0.010 * | 0.408 ± 0.039 * |
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Shaw, A.M.; Qasem, A.; Naser, S.A. Modulation of PTPN2/22 Function by Spermidine in CRISPR-Cas9-Edited T-Cells Associated with Crohn’s Disease and Rheumatoid Arthritis. Int. J. Mol. Sci. 2021, 22, 8883. https://doi.org/10.3390/ijms22168883
Shaw AM, Qasem A, Naser SA. Modulation of PTPN2/22 Function by Spermidine in CRISPR-Cas9-Edited T-Cells Associated with Crohn’s Disease and Rheumatoid Arthritis. International Journal of Molecular Sciences. 2021; 22(16):8883. https://doi.org/10.3390/ijms22168883
Chicago/Turabian StyleShaw, Ameera M., Ahmad Qasem, and Saleh A. Naser. 2021. "Modulation of PTPN2/22 Function by Spermidine in CRISPR-Cas9-Edited T-Cells Associated with Crohn’s Disease and Rheumatoid Arthritis" International Journal of Molecular Sciences 22, no. 16: 8883. https://doi.org/10.3390/ijms22168883