EAU was induced as previously described [21 (link)-23 (link)]. Briefly C57BL/6J mice were immunized subcutaneously with 500 µg of IRPB peptide 1–20 (GPTHLFQPSLVLDMAKVLLD; GL Biochem, Shanghai Ltd., China) emulsified in complete Freund’s adjuvant (CFA, H37Ra, Difco Laboratories, Detroit, MI, USA). Mice were administered with an additional intraperitoneal injection of 100 µl (1.5 µg) of Bordetella pertussis toxin (Tocris Bioscience, UK). Retinal inflammation develops at day 12-14 post-immunisation (p.i.), and peaks at day 22-25 p.i. The severity of inflammation declines after the peak stage, however, retinal inflammation remains active for over 4 months [22 (link)].
Bordetella pertussis toxin
Manufactured by Bio-Techne
Sourced in United Kingdom
Bordetella pertussis toxin is a protein product derived from the Bordetella pertussis bacteria. It is used in research applications for the study of bacterial pathogenesis and host-pathogen interactions.
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4 protocols using bordetella pertussis toxin
1
Experimental Autoimmune Uveitis Induction
2
Immunization and Intravitreal Infliximab Therapy for Experimental Autoimmune Uveitis
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Female B10.RIII mice were immunized subcutaneously in one flank with 50 μg RBP-3161–180 in phosphate buffered saline (PBS) emulsified with Complete Freund’s Adjuvant (CFA) supplemented with 1.5 mg/ml Mycobacterium tuberculosis complete H37 Ra (BD Biosciences, Oxford, UK) (1:1 vol/vol). The mice also received 1 μg Bordetella pertussis toxin (Tocris, Bristol, UK) intraperitoneally (i.p.).
For local administration of infliximab or FpFinfliximab, intravitreal injections were performed on day 10 post-immunization. In brief, the eye was proptosed and held in position with a pair of forceps, while 15 μg of infliximab or FpFinfliximab diluted in 2 μl PBS was injected using a 33-gauge hypodermic needle (Esslab, Essex, UK). The injection site was treated with chloramphenicol and globe reposited.
For local administration of infliximab or FpFinfliximab, intravitreal injections were performed on day 10 post-immunization. In brief, the eye was proptosed and held in position with a pair of forceps, while 15 μg of infliximab or FpFinfliximab diluted in 2 μl PBS was injected using a 33-gauge hypodermic needle (Esslab, Essex, UK). The injection site was treated with chloramphenicol and globe reposited.
3
Mouse Model of Experimental Autoimmune Uveitis
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On Day 0, B10.RIII mice were immunized subcutaneously with 100 μL of recombinant human interphotoreceptor retinoid binding protein (IRBP) peptide (161–180) (1 mg/mL) emulsified 1:1 (v:v) in complete Freund's adjuvant containing 2.5 mg Mycobacterium tuberculosis complete H37Ra (BD Biosciences). After immunization, each mouse was injected with 1 μg of Bordetella pertussis toxin (Tocris Bioscience) intraperitoneally. Intraperitoneal administration of IOX1 (12.5 mg/kg) or DMSO solution was performed twice daily post-immunization. At peak disease (Day 14), mice were sacrificed, and tissues collected for ex-vivo analysis. To assess the clinical score of EAU, we used either a TEFI system22 (link) or a Micron IV murine fundus camera (Phoenix Research Labs, USA) and contemporaneous Spectralis-optical coherence tomography (OCT) scanning.23 The clinical scores were given by two independent experienced observers in a blindfold manner based on the criteria for EAU scoring as described previously.22 (link),23
4
Induction of Experimental Autoimmune Uveitis in Mice
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Eight-to 12-weekeold C57BL/6J, SOCS3 fl/fl and LysM Cre/þ SOCS3 fl/fl mice (both in C57BL/6J background) were used in the study. The LysM Cre/þ SOCS3 fl/fl mice were obtained by crossbreeding the SOCS3 fl/fl mice and the LysM-Cre mice. The absence of SOCS3 in myeloid cells in the LysM Cre/þ SOCS3 fl/fl mice was confirmed by Western blotting of peritoneal macrophages. The SOCS3 fl/fl mice were used as controls in some experiments. All mice were maintained in the Biological Research Unit at Queen's University Belfast and exposed to a 12-hour light/night cycle with free access to water and food. All procedures were conducted under the regulation of the UK Home Office Animals (Scientific Procedures) Act 1986 and approved by the Animal Welfare & Ethical Review Body of Queen's University Belfast.
EAU was induced in mice using a protocol described previously. 22, 23 Briefly, mice were immunized with 500 mg of IRBP 1-20 (GPTHLFQPSLVLDMAKVLLD, GL Biochem, Shanghai, China), emulsified 1:1 in complete Freund's adjuvant (Difco Laboratories, Detroit, MI), and supplemented with 2.5 mg/mL of Mycobacterium tuberculosis H37Ra (Difco Laboratories). An additional 1 mg of Bordetella pertussis toxin (Tocris Bioscience, Bristol, UK) was administered intraperitoneally immediately after immunization injection.
EAU was induced in mice using a protocol described previously. 22, 23 Briefly, mice were immunized with 500 mg of IRBP 1-20 (GPTHLFQPSLVLDMAKVLLD, GL Biochem, Shanghai, China), emulsified 1:1 in complete Freund's adjuvant (Difco Laboratories, Detroit, MI), and supplemented with 2.5 mg/mL of Mycobacterium tuberculosis H37Ra (Difco Laboratories). An additional 1 mg of Bordetella pertussis toxin (Tocris Bioscience, Bristol, UK) was administered intraperitoneally immediately after immunization injection.
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