The Role of Interferon Regulatory Factor 1 in Regulating Microglial Activation and Retinal Inflammation
Abstract
:1. Introduction
2. Results
2.1. Increased IRF1 Protein Expression Levels in Activated Retinal Microglia
2.2. IRF1 Knockout (KO) Defers BV2 Microglial Proliferation
2.3. IRF1′s Involvement in Microglial Migration
2.4. IRF1′s Role in the Phagocytosis of Activated Microglia
2.5. IRF1-KO Alters the Gene Expression Profile of M1 vs. M2 Activation State
2.6. IRF1 KO Reduces Microglial Cytotoxicity to Retinal Cells in Cultures
2.7. IRF1 Knockdown Reduces Retinal Microglial Activation and Inflammation In Vivo
3. Discussion
4. Materials and Methods
4.1. Experimental Animals
4.2. Intravitreal Injection of LPS and siRNA in Mice
4.3. Retinal Flat Mounts and Immunofluorescent Stain
4.4. Cell Cultures and Treatments
4.5. IRF1 Knockdown (KD) and Overexpression (OE) in BV2 Microglial Cells
4.6. IRF1 Knockout (KO) from BV2 Cells with the CRISPR/Cas9 Method
4.7. Cell Live and Death Assays
4.8. Cell Migration Assay
4.9. Microglia Phagocytosis of Zymosan Bioparticles
4.10. Flow Cytometry
4.11. Real-Time Quantification PCR
4.12. Western Blots and Densitometry Analysis
4.13. Immunohistochemistry and Immunofluorescence
4.14. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Target Protein & Secondary Antibody (2 Ab) | Produced by | Catalog Number | Host | WB Dilution | IF Dilution |
---|---|---|---|---|---|
IRF1 | CST | #8478 | rabbit | 1:1000 | - |
inos | abcam | ab283655 | rabbit | - | 1:100 |
GAPDH | arigo | ARG10112 | mouse | 1:1000 | - |
actin | arigo | ARG62346 | mouse | 1:1000 | - |
Iba1 | wako | 019-19741 | rabbit | - | 1:500 |
GFAP | Thermo | 13-0300 | Rat | - | 1:100 |
DAPI | Thermo | C0060 | - | 1:5000 | |
Goat anti-Mouse IgG antibody (HRP) | arigo | ARG65350 | Goat | 1:5000 | - |
Goat anti-Rabbit IgG antibody(HRP) | arigo | ARG65351 | Goat | 1:5000 | - |
Goat anti-Mouse IgG 2 Ab, Alexa Fluor 488 | thermo | A-11001 | Goat | - | 1:500 |
Donkey anti-Rat IgG 2 Ab, Alexa Fluor Plus 488 | thermo | A48269 | Donkey | - | 1:500 |
Goat anti-Rabbit IgG 2 Ab, Cyanine3 | thermo | A10520 | Goat | - | 1:500 |
Oligo Name | Sequence |
---|---|
IRF1-siRNA-01 | GCACCACTGATCTGTATAA |
IRF1-siRNA-02 | CCAAGACATGGAAGGCAAA |
IRF1-siRNA-03 | GGACATTGGGATAGGCATA |
IRF1-sgDNA | CCATGCCAATCACTCGAATG |
CCL5-Forward | TGCCCACGTCAAGGAGTATT |
CCL5-Reverse | GCGGTTCCTTCGAGTGACA |
iNOS-Forward | GGTGAAGGGACTGAGCTGTT |
iNOS-Reverse | CGTTCTCCGTTCTCTTGCAGT |
COX2-Forward | GCGAGCTAAGAGCTTCAGGA |
COX2-Reverse | TCATACATTCCCCACGGTTT |
CCL2-Forward | TTAAAAACCTGGATCGGAACCAA |
CCL2-Reverse | GCATTAGCTTCAGATTTACGGGT |
IL-6-Forward | TAGTCCTTCCTACCCCAATTTCC |
IL-6-Reverse | TTGGTCCTTAGCCACTCCTTC |
CD47-Forward | GGTGGGAAACTACACTTGCGAAG |
CD47-Reverse | CTCCTCGTAAGAACAGGCTGATC |
Arg1-Forward | ACTGGAACCCAGAGAGAGCA |
Arg1-Reverse | ACAGACCGTGGGTTCTTCAC |
CD206-Forward | TTCAGCTATTGGACGCGAGG |
CD206-Reverse | GAATCTGACACCCAGCGGAA |
TGF-b-Forward | AGCCCGAAGCGGACTACTAT |
TGF-b-Reverse | TCCACATGTTGCTCCACACT |
Actin-Forward | AGGCGGACTGTTACTGAGC |
Actin-Reverse | CGCCTTCACCGTTCCAGTT |
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Yang, X.; Diaz, V.; Huang, H. The Role of Interferon Regulatory Factor 1 in Regulating Microglial Activation and Retinal Inflammation. Int. J. Mol. Sci. 2022, 23, 14664. https://doi.org/10.3390/ijms232314664
Yang X, Diaz V, Huang H. The Role of Interferon Regulatory Factor 1 in Regulating Microglial Activation and Retinal Inflammation. International Journal of Molecular Sciences. 2022; 23(23):14664. https://doi.org/10.3390/ijms232314664
Chicago/Turabian StyleYang, Xu, Valeria Diaz, and Hu Huang. 2022. "The Role of Interferon Regulatory Factor 1 in Regulating Microglial Activation and Retinal Inflammation" International Journal of Molecular Sciences 23, no. 23: 14664. https://doi.org/10.3390/ijms232314664