Short chain fatty acids ameliorate immune-mediated uveitis partially by altering migration of lymphocytes from the intestine
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Female C57Bl/6J and Kaede transgenic (on C57Bl/6J background) mice were immunized subcutaneously into the base of the tail and each thigh with 200 µl total of the emulsion containing IRBP1–20 or IRBP651–670 (300–500 µg peptide/animal; AnaSpec, Fremont, CA, USA; GenScript, Piscataway, NJ, USA) made in incomplete Freund’s adjuvant (IFA; Sigma-Aldrich) with heat-inactivated Mycobacterium tuberculosis antigen (5 mg/ml; BD, Franklin Lakes, NJ, USA). In addition, pertussis toxin from Bordetella pertussis (1 µg dose/ animal; Sigma-Aldrich) was injected subcutaneously once at the time of immunization per previously published reports6. Because the timing of EAU peak intraocular inflammation shifted from 3 weeks post-immunization to 16 days post-immunization depending on the IRBP supplier (AnaSpec vs. GenScript), time points were adjusted and equivalently expressed in our time course experiments. Female B10.RIII mice were immunized with 200 µl of the emulsion containing IRBP161–180 (15 µg peptide/animal; AnaSpec, Fremont, CA) made in IFA with the Mycobacterium tuberculosis antigen (2.5 mg/ml) as previously reported4. Clinical EAU scores were evaluated by fundus examination with a 90D lens (Volk, Mentor, OH, USA) and indirect ophthalmoscope (Keeler, Sacramento, CA, USA) weekly until euthanasia. Fundus photographical images were taken using an endoscope (Endoscopy Support Service, Brewster, NY, USA) and Nikon D3200 camera (Melville, NY, USA). |
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