Blimp-1 and c-Maf regulate common and unique immune gene networks to protect against distinct pathways of pathobiont-induced colitis [ATAC-Seq]

Using single-cell RNA-seq of colon lamina propria leukocytes (LPL), together with ATAC-seq, ChIP-seq and RNA-seq from colon LPL and sorted CD4+ T from colon lamina propria during oral infection with the pathobiont Helicobacter hepaticus we show that Blimp-1 and c-Maf both positively regulate Il10 gene expression, but also cooperate to negatively regulate a large network of proinflammatory effector genes in T cells. While T cell-specific deletion of either Prdm1, Maf or both transcription factors did not result in overt inflammation of the colon at steady state, H. hepaticus infection resulted in mild/moderate colitis in both the Prdm1fl/flCd4Cre and Maffl/flCd4Cre single T cell-specific transcription factor deficient mice, while the double knockout Prdm1fl/flMaffl/flCd4Cre infected mice suffered severe colitis and showed a massive increase in T cell effector genes in colonic LPL. Blimp-1 and c-Maf cooperate to negatively regulate genes including Ifng, Csf2, Il23r, Stat4, Il18r1 and Il2rg, and deletion of both these transcription factors in T cells translated to an exacerbated pathogenic TH1, IFN-g+GM-CSF+ effector cell response. c-Maf, additionally negatively regulated Il17a expression which was elevated in the LPL as assessed by bulk RNA-seq and scRNA-Seq analysis in the H. hepaticus infected Maffl/flCd4Cre mice. Overall design: ATAC-seq of colon lamina propria CD4+ T cells isolated from mice with Cd4Cre-mediated deletion of either Prdm1, Maf or both Prdm1 Maf on Day 14 following infection with Helicobacter hepaticus

我们通过结肠固有层白细胞（colon lamina propria leukocytes, LPL）的单细胞RNA测序（single-cell RNA-seq），结合口服感染致病共生菌肝螺杆菌（Helicobacter hepaticus）期间，结肠LPL及分选获得的结肠固有层CD4+ T细胞的ATAC-seq、ChIP-seq与RNA-seq数据，证实转录因子Blimp-1与c-Maf均可正向调控Il10基因的表达，且二者协同负向调控T细胞内庞大的促炎效应基因网络。尽管稳态条件下，特异性敲除T细胞内的Prdm1、Maf或二者之一均未引发明显的结肠炎症，但肝螺杆菌感染后，Prdm1fl/flCd4Cre与Maffl/flCd4Cre两种单T细胞特异性转录因子缺陷小鼠均出现轻中度结肠炎；而双敲除Prdm1与Maf的Prdm1fl/flMaffl/flCd4Cre感染小鼠则罹患重度结肠炎，且结肠LPL内T细胞效应基因出现显著上调。Blimp-1与c-Maf可协同负向调控包括Ifng、Csf2、Il23r、Stat4、Il18r1及Il2rg在内的多个基因；T细胞内这两种转录因子的双缺失会加剧致病性TH1型IFN-γ+GM-CSF+效应细胞应答。此外，c-Maf可负向调控Il17a的表达；在肝螺杆菌感染的Maffl/flCd4Cre小鼠中，通过批量RNA测序（bulk RNA-seq）与单细胞RNA-seq分析可见，其LPL内Il17a的表达水平显著升高。本研究整体实验设计如下：于肝螺杆菌感染第14天，分离经Cd4Cre介导敲除Prdm1、Maf或二者同时敲除的小鼠结肠固有层CD4+ T细胞，开展ATAC-seq检测。