RNA-seq analysis of murine experimental autoimmune thyroiditis (EAT) following modulation of tryptophan-kynurenine pathway

Female C57BL/6 mice (6-8 weeks old) were used to establish the model. After 1-week adaptive feeding, they were randomly divided into 4 groups (n=10 per group).Control (Con) group: From the second week onward, the mice received 0.05% NaCl as drinking water. On the same week, they were given multiple subcutaneous injections of PBS at the dorsal, abdominal, and cervical regions, and daily intraperitoneal PBS injections were initiated and continued until the end of the experiment. Two weeks later, multi-site subcutaneous PBS injections were repeated.HT model (HT) group: Beginning in the second week, the mice received 0.05% NaI as drinking water and were administered the first series of subcutaneous injections of porcine thyroglobulin (200 ug per mouse) emulsified in complete Freund's adjuvant (CFA) at multiple sites on the dorsal, abdominal, and cervical regions. Concurrently, daily intraperitoneal injections of PBS were initiated and maintained until the end of the experiment. Two weeks later, a second set of subcutaneous injections of porcine thyroglobulin (200 ug per mouse) emulsified in incomplete Freund's adjuvant (IFA) was given at multiple sites.HT + Trp (HT+T) group: The regimen was identical to that of the HT group, except that the daily intraperitoneal injection was replaced with 20 mg/kg Trp (dissolved in PBS).HT + Kyn-pathway inhibitor (HT+I) group: The regimen was identical to that of the HT group, except that the daily intraperitoneal PBS injection was replaced with 20 mg/kg of the Kyn-pathway inhibitor (IDO1/TDO-IN-4) administered every other day. IDO1/TDO-IN-4 is a potent dual inhibitor of both indoleamine IDO1 and TDO and therefore functions as an effective Kyn-pathway inhibitor. A stock solution of 20 mg/ml was prepared in DMSO and diluted ten-fold with 20% (w/v) SBE-beta-CD in normal saline immediately before use.Four weeks after the final immunization challenge, the mice were euthanized, and thyroid glands were collected for RNA sequencing.