Transcriptional profilling of In vitro-primed C.rodentium-specific WT or Caspase-1 deficient Th cells. Transcriptional profilling of In vitro-primed C.rodentium-specific WT or Caspase-1 deficient Th cells

Introduction: We reported that caspase-1 is highly expressed in in vitro-primed C.rodentium-specific Th cells. Caspase-1 deficient (Casp1d10) naïve T cells differentiate into pathogen-specific Th17 cells at an suboptimal level and fail to protect against C.rodentium infection. To gain insight into the regulatory pathways exerted by caspase-1 in Th cells, we performed RNA-seq on in vitro-primed WT and Casp1d10 Th cells. Method: We stimulated WT C57/BL6J splenic CD11c+ DCs with 10ug/ml C.rodentium lysate for 5 hours. Then DCs are washed and co-cultured in 1:5 ratio with naïve WT C57/BL6J or Casp1d10 CD4 T cells for 10 days. CFSE-CD90+ (pathogen-specific Th cells) were FACS-sorted and subjected to mRNA-seq analysis. Conclusion: We found that WT Cr-specific Th cells have higher expression of Th17 genes, including Il17a,f,Il22 and Rorc. Casp1Δ10 Cr-specific Th cells exhibited higher expression of iNOS genes and exogenous lipid metabolism genes such as Cd36, suggesting a distinct cellular metabolism potentially regulated by Caspase-1. Overall design: mRNA profiles of WT and Casp1d10 Cr-specific Th cells.

研究背景：本研究团队此前报道，半胱天冬酶-1（caspase-1）在体外预激活的啮齿类柠檬酸杆菌（*Citrobacter rodentium*, C. rodentium）特异性T辅助细胞（Th细胞）中高表达。半胱天冬酶-1缺陷型（Casp1Δ10）初始T细胞分化为病原体特异性Th17细胞的水平处于亚最优状态，且无法抵御啮齿类柠檬酸杆菌感染。为深入解析半胱天冬酶-1在Th细胞中发挥调控作用的信号通路，本研究对体外预激活的野生型（Wild Type, WT）及Casp1Δ10 Th细胞开展了RNA-seq测序分析。

实验方法：本研究以10 μg/ml的啮齿类柠檬酸杆菌裂解物刺激野生型C57BL/6J小鼠脾脏来源的CD11c阳性树突状细胞（CD11c+ DCs），刺激时长为5小时。随后洗涤树突状细胞，并按照1:5的比例与初始型野生型C57BL/6J或Casp1Δ10 CD4阳性T细胞共培养10天。经羧基荧光素二醋酸盐琥珀酰亚胺酯（CFSE）标记且CD90阳性的病原体特异性Th细胞通过荧光激活细胞分选术（FACS）纯化后，进行mRNA测序分析。

研究结论：本研究发现，野生型啮齿类柠檬酸杆菌特异性Th细胞的Th17相关基因（包括Il17a、Il17f、Il22及Rorc）表达水平更高。而Casp1Δ10啮齿类柠檬酸杆菌特异性Th细胞的诱导型一氧化氮合酶（iNOS）相关基因及外源性脂质代谢相关基因（如Cd36）表达水平显著升高，提示半胱天冬酶-1可能调控了一条独特的细胞代谢通路。

实验整体设计：野生型与Casp1Δ10啮齿类柠檬酸杆菌特异性Th细胞的mRNA表达谱。