TH17 cells contribute to uveitis and scleritis and are inhibited by IL-27/STAT1 in the retina (2) Expression of IL-17 in mouse PBMC, lymph node and retina
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
Expression of IL-17 in mouse PBMC, lymph node and retina is temporally correlated with progression of experimental autoimmune uveoretinitis (EAU)
Uveitis is a diverse group of intraocular inflammatory diseases that cause severe visual loss and morbidity and is characterized by inflammatory attack of the uvea as well as the neuroretina 1. The disease may be of infectious or putative autoimmune etiology. The latter include birdshot retinochoroidopathy, sympathetic ophthalmia, Behçet’s disease, Vogt-Koyanagi-Harada syndrome, and ocular sarcoidosis 1. Understanding the immunopathogenic mechanisms of uveitis has benefited enormously by the development of an animal model of uveitis, experimental autoimmune uveitis (EAU). EAU is a predominantly a T cell-mediated intraocular inflammatory disease induced in susceptible species by active immunization with ocular-specific proteins (or peptides derived from them) and is transferable to naive syngeneic animals by injection of in vitro-activated CD4+, MHC class II-restricted T cell lines specific to retinal Ags 2-4. The two major uveitogenic retinal proteins are S-Ag (also termed “arrestin”) and interphotoreceptor retinoid-binding protein (IRBP) 2-4. In this study, we have used the EAU mouse model to further establish that TH17 cells are involved in pathogenic mechanisms of uveitis by showing that EAU can be ameliorated by treatment with antibody to IL-17.
The procedure consists of the following steps:
Active induction of experimental autoimmune uveoretinitis (EAU)
Preparation of antigen/CFA emulsion
Immunization
Clinical grading of mice with EAU by fundoscopy
Preparation of sections for ocular histology
Inhibition of EAU by IL-17 neutralizing antibody
IL-27 expression in PBMC or retina is temporally correlated with progression of EAU
Other protocols related to our Nature Medicine paper can be found here:
Detection of TH17 cells in human blood
Materials
Reagents
• B10.RIII and C57BL/6 mice (6-8 weeks old) (Jackson Laboratory).
• Interphotoreceptor retinoid-binding protein (IRBP) proteins are prepared from bovine retina by Concanavalin A (Con-A) Sepharose affinity chromatography and fast performance liquid chromatography (FPLC) as previously described 5
• Human IRBP peptide 1-20 (GPTHLFQPSLVLDMAKVLLD) and 161-180 (SGIPYIISYLHP GNTILHVD) was synthesized on an Applied Biosystems 432A Peptide Synthesizer using Fmoc Chemistry or purchased from Ana Spec Inc.
• Complete Freund adjuvant (CFA)
• Mycobacterium tuberculosis strain H37Ra (Sigma).
• Bordetella pertussis toxin (PTX) (Sigma).
• IL-17 antibodies (R&D, clone 50104.11).
• IgD2a isotype control antibody, (R&D, clone 54447.11).
• Human IRBP peptide (1-20).
• Mixture of Ketamine (100 mg/ml) and Xylasine hydrochloride (“Rompan”) as anesthetic drug in animal using protocols.
• 1% Mydriacyl (tropicamide ophthalmic solution), USP (Alcon).
• Dacriose (Sterile eye irrigating solution) (Johnson & Johnson).
• Ak-dilate (Akorn).
• Tears Renewed (lubricant Eye drop) (Alcon)
• 10% formaldehyde in PBS.
Equipment
• Sonicator equipped with microtip probe.
• 15 ml and 50 ml polypropylene tubes
• 1 ml syringe
• 19-G blunt-tipped and 25-G needles.
• Cover glass 22×22 0.13
• Small dissecting scissors and forceps.
• CO2 Rodent Euthanasia chamber.
• Binocular dissecting microscope with coaxial illumination
Procedure
Active induction of experimental autoimmune uveoretinitis (EAU)
EAU was induced with 150µg bovine IRBP and 300µg human IRBP peptide (1-20) in C57bl/6 strain. Disease was induced in B10RIII mice with 150µg bovine IRBP and 12-20 µg human IRBP peptide (161-180) and clinical disease in both strains were established by fundoscopy and histology. Initial signs EAU is observed approximately 7-12 days after immunization, with full-blown disease developing by post-immunization day-14. By day-21 post-immunization there is substantial reduction of inflammatory cells and disease resolution characterized by absence of inflammatory cells or inflammatory cytokines in the retina occurs 28 days post-immunization6.
Preparation of antigen/CFA emulsion
1| Into a 50 ml polypropylene tube, add 0.5 ml stock bovine IRBP protein (6 mg/ml) and 6 mg lyophilized human IRBP peptide (1-20) and bring mixture to a final volume of 2 ml
2| Add an equal volume or 2 ml of Complete Freund adjuvant (CFA) containing Mycobacterium tuberculosis strain H37Ra (2.5 mg/ml), mix and chill on ice for 10 min
3| Sonicate at high speed on ice for 20 seconds followed by 30 seconds intervals
4| Repeat 4-5 times until the mixture appears as a thick white cream.
5| Centrifuge at 1200 RPM for 1 min.
6| Prepare CFA emulsion without IRBP to serve as control.
7| Transfer emulsion into in 1 ml syringes with 19 gauge blunt-ended needles
8| Prepare PTX (2 ug/ml) solution in 1 ml syringes with 25 gauge needles
Immunization
1| Prepare sufficient PTX (2 ug/ml) solution (100µl/mouse)
2| Prepare sufficient CFA/Antigen emulsions (200µl/mouse)
3| Before injections expel any air bubble in the syringe containing PTX or emulsion
4| Inject each mouse with PTX solution (100 µl) intraperitoneally
5| Inject emulsion subcutaneously into base of tail (100 µl) and each thigh (50 µl)
6| Sacrifice the immunized at post-immunization day-7, -14, -21, and -28.
Controls: Un-immunized WT mice; mice immunized with CFA emulsion (without IRBP)
Clinical grading of mice with EAU by fundoscopy
1| Restrain mice by anethesia: intraperitoneal injection of mixture of Ketamine and xylazine (Rompun) at 30 mg/kg and 9 mg/kg, respectively.
2| After 5-10 min, dilate pupils by applying 1 drop of Mydriacyl (1%) to each eye.
3| Gently press the cover glass on the cornea.
4| Adjust head position to focus retina to the fundoscope for examination
Note: Mydriacyl causes temporary lens opacification within 15-20 min after application; it is important to complete examination within this time.
Preparation of sections for ocular histology
1| Incubate enucleated eyes overnight in 10% buffered formaldehyde
2| Embed eyes and cut 5µM paraffin sections in anterior/posterior orientation.
3| Stain sections in hematoxylin & eosin (H&E).
4| Grading of EAU pathology is as previously described ().
Inhibition of EAU by IL-17 neutralizing antibody
1| Active immunization of C57BL/6 mice with IRBP in CFA
2| Dilute IL-17 or IgD2a isotype control antibodies in PBS
3| Inject 500 µl IL-17 antibody (350 µg) intraperitoreally into treatment group (4 mice) 1 day before immunization with IRBP/CFA emulsion (day -1).
4| Inject 500 µl IgD2a isotype control antibody (350 µg) intraperitoreally into control group (4 mice) 1 day before immunization with IRBP/CFA emulsion (day -1).
5| Repeat Ab injection every other day until day 17 post-immunization.
6| Perform fundscopic examinations at post-immunization days 12, 14 and 16.
7| Sacrifice mice at post-immunization day-19 and perform histology
IL-27 expression in PBMC or retina is temporally correlated with progression of EAU
1| Immunise mice (8) with CFA or IRBP/CFA emulsion
2| Monitor EAU progression by fundoscopy and histology
3| Sacrifice mice at post-immunization days, 0, 7, 14, 21 and 28
4| Section 1 eye for histology and stain with H&E
5| Isolate RNA and protein extracts from retina, PBMC or lymph nodes
6| Analyze IL-27 protein expression by Western blotting
7| Analyze IL27 and Ebi3 expression by RT-PCR and qPCR.
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