Sepsis and severe surgical conditions cause profound decrease in epithelial cell proliferation in the intestinal crypts. This pathophysiological response disrupts intestinal epithelial homeostasis, which is thought to contribute to the pathogenesis of sepsis-induced immune dysfunction and multiple organ failure. Evidence shows that sepsis is associated with increased levels of interferon ? (IFN?), a proinflammatory cytokine known to inhibit intestinal epithelial cell (IEC) proliferation in vitro. In preliminary studies, we found that IFN? plays an important role in disruption of crypt IEC proliferation in sepsis. The proliferative cells in the intestinal crypts are composed of Lgr5-expressing crypt base columnar cells (Lgr5+-CBCs, a group of intestinal stem cells [ISCs]) and transit amplifying cells (TACs). However, the mechanism by which sepsis-activated IFN? signal affects proliferation of these cells has not been rigorously studied. Furthermore, little is known about how to sustain proliferation of Lgr5+-CBCs and TACs to maintain homeostasis in sepsis. Recently, we found that sepsis is associated with de novo expression of a long noncoding RNA (lncRNA) molecule, H19, in Lgr5+-CBCs and TACs in the intestinal crypts. Remarkably, we discovered that H19 lncRNA plays a critical role in attenuating sepsis-induced reduction of crypt IEC proliferation and promoting intestinal epithelial regeneration. Mechanistically, H19 lncRNA binds to molecules that inhibit cell proliferation and attenuates their activity. Collectively, these data suggest that IFN? signaling and H19 lncRNA play opposing roles in the regulation of Lgr5+-CBC and TAC proliferation in sepsis. In this project, we will test the hypothesis that sepsis-induced inflammation activates de novo expression of H19 lncRNA, which in turn antagonizes the deleterious effect of the IFN? signal axis, thereby releasing inhibition of Lgr5+-CBC and TAC proliferation and rescuing homeostasis of the intestinal mucosa. To achieve this goal, we will execute three complementary aims: (1) We will examine whether and how septic inflammation dysregulates proliferation of Lgr5+-CBCs and TACs by focusing on the role of IFN? signaling. We will precisely study the harmful effect of sepsis on proliferation of Lgr5+-CBCs and TACs in vivo utilizing a clinically relevant mouse model of polymicrobial sepsis, a novel method that combines 5-ethynyl-2'-deoxyuridine labeling with multi-probe fluorescence in situ hybridization assay, and flow cytometry and cell sorting technology. Furthermore, we will delineate how IFN? signaling leads to inhibition of Lgr5+-CBC and TAC proliferation in sepsis. (2) We will investigate the molecular mechanisms by which de novo expressed H19 lncRNA antagonizes the inhibitory effect of sepsis on Lgr5+-CBC and TAC proliferation, taking a multidisciplinary in vivo and in vitro approach that incorporates cell biology, organoid culture, molecular biology, and mouse genetic engineering techniques. (3) We will study how septic inflammation induces de novo expression of H19 lncRNA in IECs. Successful achievement of these aims will fill gaps in knowledge about the regulation of intestinal epithelial renewal in sepsis and ultimately lead to development of new therapies.

Public Health Relevance

Intestinal epithelial homeostasis is dysregulated during systemic inflammation associated with sepsis and critical surgical conditions such as severe burn and trauma, which contributes to impairment of the immune system and vital organ function. In this project, we will address the molecular mechanisms by which septic inflammation alters intestinal stem cell and progenitor cell proliferation, an important event for maintaining intestinal epithelial homeostasis. This work will lead to the identification of novel molecular targets and will ultimately advance strategies for restoring intestinal epithelial homeostasis in patients with sepsis and critical surgical illness.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Surgery, Anesthesiology and Trauma Study Section (SAT)
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Greenwel, Patricia
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Children's Memorial Hospital (Chicago)
United States
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