TH17 cells contribute to uveitis and scleritis and are inhibited by IL-27/STAT1 in the retina (5) Chromatin immunoprecipitation
Lab/Group: Egwuagu Lab (NIH)
** An editorial error was made: Cheng-Rong Yu prepared this manuscript and should have been the first author.
Related Journal & Article Information
Journal: Nature Medicine
Article Title: TH17 cells are expanded by IL-2 during Uveitis or Scleritis and inhibited by IL-27/STAT1 pathways
Introduction
This protocol is for Chromatin Immunoprecipitation.
Other protocols related to our Nature Medicine paper can be found here:
Detection of TH17 cells in human blood
Expression of IL-17 in mouse PBMC, lymph node and retina
Materials
Reagents
EZ ChipTM chromatin immunoprecipitation kit (Upstate Biotechnology, Charlottesville, VA)
• Human retinal pigment epithelium cell line (ARPE-19)
• 37% Formaldehyde
• Molecular Biology grade Proteinase K
• Glycogen or tRNA
• 50% Phenol/50%Chloroform (containing 1% isoamyl alcohol)
• Protein G agarose beads (Upstate Biotechnology)
• Protein A Agarose/Salmon sperm DNA (Upstate, Catalog #16-157C)
• Mouse Stat1-specific antibody (Santa Cruz Biotechnology, Santa Cruz, CA)
• IL-27 promoter site-specific primers.
• Solutions
ChIP Dilution Buffer: 0.01% SDS, 1.1% Triton X- 100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl (catalog # 20-153).
Elution buffer: 1% SDS, 0.1 M NaHCO3
Low Salt Immune Complex Wash Buffer: 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl. (catalog # 20-154).
High Salt Immune Complex Wash Buffer: 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl (catalog # 20-155)
LiCl Immune Complex Wash Buffer: 0.25 M LiCl, 1% IGEPAL-CA630, 1% deoxycholic acid (sodium salt), 1 mM EDTA, 10 mM Tris, pH 8.1 (catalog # 20-156).
SDS Lysis Buffer: 10 mL 1% SDS, 10 mM EDTA, 50 mM Tris, pH 8.1 (catalog # 20-163).
Equipment
• Sonicator
Procedure
1| Crosslink protein/DNA by adding 37% (54 µl) formaldehyde to 2ml of media
2| Shake gently to mix and then incubate at room temperature for 10 min.
3| Add 10X Glycine solution (200 µl) to quench unreacted formaldehyde
4| Mix gently and incubate at room temperature for 5 min.
5| Remove medium and wash with cold PBS (2X)
6| Add 0.1ml PBS containing protease inhibitor cocktail
7| Collect cells, centrifuge at 700xg for 3 min at 4 oC and discard supernatant
8| Resuspend cell pellet in 200μl lysis buffer containing protease inhibitor cocktail.
9| Sonicate cell lysate on ice for 4 second 3 times
10| Centrifuge at 14,000 rpm at 4 0C for 10 min and transfer supernatant (~100 μl) to fresh tube
11| Add 900 μl of dilution buffer containing protease inhibitor cocktail
12| Add 60 μl of protein G agarose and incubate for 1 hr at 4 0C with rotation
13| Centrifuge at 3000g for 1 min and collect supernatant into fresh tube
14| Add 4μg of anti STAT1 antibody and incubate overnight at 4 0C with rotation
15| Add 60 μl of protein G agarose for 1 hr at 4 0C with rotation
16| Centrifuge at 3000X g for 1 min and remove supernatant
17| Wash pellet with low salt wash buffer, high salt wash buffer, LiCl immune complex wash buffer and finally TE buffer
18| Add 100 μl of elution buffer and mix gently
19| Incubate at room temperature for 15 min
20| Centrifuge at 3000 x g for 1 min and collect supernatant into new tube
21| Repeat step 19 to 21 and combine supernatant
22| Add 8ul 5M NaCl and incubate at 65 oC for 5 hrs
23| Add 1ul of RNase A and incubate for 30 min at 37 oC
24| Add 4ul 0.5M EDTA, 8ul 1M Tris-HCl and 1ul Proteinase K and incubate at 45oC for 2 hrs
25| Add 1ml of bind reagent A to each tube and mix well
26| Transfer 600 μl of mixture to spin filter in collection tube and centrifuge at 14,000 g for 30 second
27| Discard liquid in collection tube and put the spin filter back into same collection tube
28| Transfer the remaining 600 μl of mixture to spin filter and centrifuge at 14,000 g for 30 second
29| Remove the liquid in collection tube and add 500 μl of wash reagent B to the spin filter in collection tube
30| Put the spin filter into new collection tube and add 50 μl of elution buffer to the spin filter
31| Centrifuge at 14,000 x g for 30 second and save eluate for PCR
Troubleshooting
Critical Steps
Anticipated Results
References
1. Nussenblatt, R.B. Proctor Lecture. Experimental autoimmune uveitis: mechanisms of disease and clinical therapeutic indications. Invest Ophthalmol Vis Sci 32, 3131-3141 (1991).
2. Gregerson, D.S., Obritsch, W.F., Fling, S.P. & Cameron, J.D. S-antigen-specific rat T cell lines recognize peptide fragments of S-antigen and mediate experimental autoimmune uveoretinitis and pinealitis. Journal of immunology (Baltimore, Md.) 136, 2875-2882 (1986).
3. Wacker, W.B., Donoso, L.A., Kalsow, C.M., Yankeelov, J.A., Jr. & Organisciak, D.T. Experimental allergic uveitis. Isolation, characterization, and localization of a soluble uveitopathogenic antigen from bovine retina. Journal of immunology (Baltimore, Md.) 119, 1949-1958 (1977).
4. Egwuagu, C.E., Mahdi, R.M., Nussenblatt, R.B., Gery, I. & Caspi, R.R. Evidence for selective accumulation of V beta 8+ T lymphocytes in experimental autoimmune uveoretinitis induced with two different retinal antigens. J Immunol 151, 1627-1636. (1993).
5. Pepperberg, D.R., Okajima, T.L., Ripps, H., Chader, G.J. & Wiggert, B. Functional properties of interphotoreceptor retinoid-binding protein. Photochem Photobiol 54, 1057-1060 (1991).
6. Takase, H., et al. Induction of suppressors of cytokine signaling (SOCS) in the retina during experimental autoimmune uveitis (EAU): potential neuroprotective role of SOCS proteins. J Neuroimmunol 168, 118-127 (2005).
7. Yu, C.R., et al. Suppressor of cytokine signaling 3 regulates proliferation and activation of T-helper cells. J Biol Chem 278, 29752-29759 (2003).
8. Egwuagu, C.E., et al. Suppressors of cytokine signaling proteins are differentially expressed in Th1 and Th2 cells: implications for Th cell lineage commitment and maintenance. J Immunol 168, 3181-3187. (2002).
Acknowledgements
This research was funded by the Intramural Research Programs of the NEI and NIH. Authors thank Dr. Wei Zhu (Laboratory of Immunology, NEI, NIH) for assistance with fundoscopy.
Keywords
experimental autoimmune uveoretinitis (EAU); IL-27p28 immunohistochemistry and confocal microscopy; Intracellular cytokine staining assay; Retinal organ culture