Surgical injury depletes cecal crypts of their microbiota and is associated with pathoadaptive activation of stem cells during gut-derived sepsis
Author(s):
Alexander Zaborin, The University of Chicago; Jennifer DeFazio, University of Chicago; SANJIV HYOJU, University of Chicago; Monika Krezalek, University of Chicago; Vytas Bindokas, University of Chicago; Olga Zaborina, University of Chicago; John Alverdy, University of Chicago
Background: The normal microbiota provide tonic stimulation to the host immune system to protect against sepsis. The spatial context and mechanism of this action remains unknown.
Hypothesis: Here we hypothesized that loss of the normal microbiota within cecal crypts during surgical injury causes a defect in epithelial regeneration creating a high risk environment for the development of gut-derived sepsis.
Methods: We used a mouse model of gut-derived sepsis in which mice undergo a short period of pre-op starvation (H20 only), antibiotic administration (IM cefoxitin) and a 30% hepatectomy with intracecal injection of a 4 pathogen community to mimic major surgical intervention with exposure to hospital pathogens. This prep results in >50% mortality rate due to sepsis and a near 100% rescue when a fecal transplant is administered. To examine the spatial location of microbiota and pathogens, cecal crypts were analyzed by scanning electron microscopy and fluorescent in situ hybridization. In situ hybridization was used to probe for Lgr5, a biomarker of epithelial stem cells.
Results: In this multiple hit model of surgical injury we observed complete emptying of cecal crypts of their microbiota. This was not observed in mice starved alone, given antibiotics alone or both. When hepatectomy alone was performed, spotty areas of crypts emptying of the microbiota were observed. In mice undergoing the multiple hit model (starvation+antibiotic+hepatectomy), the expression of Lgr5 was highly activated and Lgr5+ cells were abnormally distributed along the villous tips of empty crypts (n=2000 crypts/group, p<0.001). In this group, the introduced pathogen community then occupied the empty crypts which resulted in a significant increase in TUNEL+ cells (n=2000 crypts/group, p<0.001). Mice administered a fecal transplant at 24 hrs following the introduction of the pathogen community were protected against mortality in association with microbiota re-colonizing the crypts (n=5 mice/group, 100 crypts/mice). The normalization of Lgr5 expression and localization was reversed in mice displaying re-colonization of their crypts by microbiota (n=2000 crypts/group, p<0.01).
Conclusions: The combination of short-term starvation, surgical injury and antibiotic use eliminates the normal microbiota in intestinal crypts providing opportunity for hospital pathogens to occupy these critical niches. The consequence of this process is a disruption of epithelial stem cell function which may have far reaching implications on host immune homeostasis.