Bacterial ADP-heptose initiates a revival stem cell program in the intestinal epithelium

The intestinal epithelium has an exceptional capacity to repair following injury, and recent evidence has suggested that YAP-dependent signaling was crucial for the expansion of Clu+ revival stem cells (revSCs) with fetal-like characteristics, which are essential for epithelial regeneration. However, neither the mechanism underlying where these revSCs emerge from nor the nature of the physiological cues that induce this revSC program, are clearly identified. Here, we first demonstrate that Alpk1 and Tifa, which encode the proteins essential for the detection of the bacterial metabolite ADP-heptose (ADP-Hep), were expressed by the stem cell pool in the intestinal epithelium. Treatment of intestinal organoids with ADP-Hep not only induced acute NF-κB pro-inflammatory signaling but also TNF-dependent apoptosis within the crypt, causing blunted proliferation and acute disruption of the crypt architecture, while also triggering induction of a revSC program. To identify the molecular underpinnings of this process, we performed single-cell RNA-seq analysis of ADP-Hep-treated organoids as well as lineage-tracing experiments. Our data reveal that ADP-Hep induced the specific ablation of the homeostatic intestinal stem cell (ISC) pool. Removal of ADP-Hep resulted in the rapid recovery of ISCs through dedifferentiation of Paneth cells, which transiently acquired revSC features and expressed nuclear YAP. Moreover, lineage tracing from Lyz1+ Paneth cells showed that ADP-Hep triggered Paneth cell de-differentiation towards pluripotent and proliferative cells in organoids. In vivo, revSC emergence in response to irradiation-induced injury was severely blunted in Tifa-deficient mice, suggesting that efficient epithelial regeneration in this model required detection of microbiota-derived ADP-Hep by the ALPK1-TIFA pathway. Together, our work reveals that Paneth cells can serve as the cell of origin for revSC induction in the physiological context of microbial stimulation, and that the transient loss of Alpk1-expressing ISCs is the initiating event for this regenerative process. ### Competing Interest Statement The authors have declared no competing interest.