Abstract
Enterotoxigenic Escherichia coli (ETEC) is a major diarrheal pathogen in children in low- to middle-income countries. Previous studies identified heat-stable enterotoxin (ST)-producing ETEC as a prevalent diarrheal pathogen in children younger than 5 years. While many studies have evaluated the interaction of ETEC heat-labile enterotoxin (LT) with host epithelium and immunity, few investigations have attempted similar studies with ST. To further understand ST pathogenesis, we examined the impact of ST on cGMP localization, epithelial cell cytokine production, and antibody development following immunization. In addition to robust intracellular cGMP in T84 cells in the presence of phosphodiesterase inhibitors (PDEis) that prevent the breakdown of cyclic nucleotides, we found that prolonged ST intoxication induced extracellular cGMP accumulation in the presence or absence of PDEis. Further, ST intoxication induced luminal cGMP in vivo in mice, suggesting that secreted cGMP may have other cellular functions. Using transcriptome sequencing (RNA-seq) and quantitative PCR (qPCR), we demonstrated that ST intoxication, or treatment with the clinically used ST mimic linaclotide, altered inflammatory cytokine gene expression, including the interleukin 1 (IL-1) family member IL-33, which could also be induced by cell-permeative 8-Br-cGMP. Finally, when present during immunization, ST suppressed induction of antibodies to specific antigens. In conclusion, our studies indicate that ST modulates epithelial cell physiology and the interplay between the epithelial and immune compartments.
Highlights
Enterotoxigenic Escherichia coli (ETEC) is a major diarrheal pathogen in children in low- to middle-income countries
Confluent T84 intestinal epithelial cell monolayers were pretreated with the phosphodiesterase inhibitors (PDEis) zardaverine and vardenafil to prevent the degradation of cGMP for 1 h before stable enterotoxin (ST) intoxication over a dose range (0, 2, 5, 10, 25, 50, and 100 ng) for 2, 6, 24, and 48 h
Despite the addition of PDEis, intracellular cGMP levels decreased over time while secretory cGMP levels increased over time, suggesting that T84 intestinal epithelial cells may make a concerted effort to secrete cGMP upon ST intoxication (Fig. 1C)
Summary
Enterotoxigenic Escherichia coli (ETEC) is a major diarrheal pathogen in children in low- to middle-income countries. To further understand ST pathogenesis, we examined the impact of ST on cGMP localization, epithelial cell cytokine production, and antibody development following immunization. Using transcriptome sequencing (RNA-seq) and quantitative PCR (qPCR), we demonstrated that ST intoxication, or treatment with the clinically used ST mimic linaclotide, altered inflammatory cytokine gene expression, including the interleukin 1 (IL-1) family member IL-33, which could be induced by cell-permeative 8-Br-cGMP. ST induced both apical and basolateral secretion of cGMP in polarized enteroid monolayers, suggesting that ST intoxication could affect interactions between the epithelial and immune compartments via cGMP [11]. It has been shown that ST reprograms epithelial signaling and that the inflammatory cytokines induced by LT in animals are damped in the presence of ST [3], interactions that could alter mucosal immune system function. During heterologous ETEC challenge studies, the majority of volunteers who developed anti-LT serological responses following LT1 ST1-ETEC B7A exposure were still susceptible to LT1ETEC E2528-C1-mediated challenge [14], supporting the idea that ST hinders development of sustained anti-LT immune or anti-ETEC responses or that anti-LT responses alone are not sufficient for long-term protection
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