ST competes with the endogenous peptides guanylin and uroguanylin for activation of the particulate guanylyl cyclase C (GC-C) receptor to induce cGMP production. 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. (ETEC) is a significant global cause of secretory diarrhea against which there are currently no licensed vaccines. Most ETEC cases occur in children younger than 5 years in low- and middle-income countries (LMICs) and in travelers and military personnel deployed to PDLIM3 LMICs and other regions where ETEC is endemic (1). ETEC isolates are classified by their enterotoxin expression profiles based on the types and combinations of enterotoxins they produce: heat-labile enterotoxin-producing (LT+) ETEC, heat-stable enterotoxin-producing (ST+; human [STh] or porcine [STp]) ETEC, and LT+ ST+ ETEC. ETEC producing any combination of these toxins can cause secretory diarrhea in humans (2). LT is a highly immunogenic protein able to induce antibodies following natural LT+-ETEC infection. LT binds to GM1 gangliosides on intestinal epithelial or immune cell surfaces and is internalized to modulate cyclic AMP (cAMP) signaling pathways. Numerous studies have focused on LT-mediated modulation of the host epithelium and immune system. LT induces expression of interleukin 1 (IL-1) family members, including IL-1 from murine jejunum and ileum (3) and IL-1 and IL-1 from murine dendritic cells (4), and potent T helper cell type 17 (TH17) and mucosal IgA responses (5, 6). In comparison, ST-specific pathogenesis remains relatively understudied despite the majority of the moderate to severe diarrheal cases resulting from ST+-ETEC or ST+ LT+-ETEC infection (2). ST is a small secreted peptide that fails to induce robust antibodies during natural ST+-ETEC exposure. ST competes with the endogenous peptides guanylin and uroguanylin for activation of the particulate guanylyl cyclase C (GC-C) receptor to induce cGMP production. ST-induced cGMP levels result in cGMP-dependent kinase C type II (cGKII) and, as with LT, protein kinase A SJFα (PKA) phosphorylation to activate CFTR (cystic fibrosis transmembrane conductance regulator) and inhibit NHE3 (sodium-hydrogen exchanger 3), resulting in secretory diarrhea (7). In fact, the ST analog linaclotide is used clinically to treat irritable bowel syndrome with symptoms of constipation (8). Activating mutations in GC-C in cases of familial diarrhea syndrome (FGDS) (9) as well as decreased expression of GC-C in ulcerative colitis SJFα (UC) (10) suggests that precise GC-C signaling is important to maintain intestinal health and homeostasis. ST applied in a continuous flow to flexible jejunal monolayers induces cGMP in secreted but not intracellular fractions, suggesting the emergence SJFα of a role for secreted cGMP in ETEC pathogenesis. Additionally, ST induced both apical and basolateral secretion of cGMP in polarized enteroid SJFα monolayers, suggesting that ST intoxication could affect interactions between the epithelial and immune compartments via cGMP (11). In fact, small cyclic nucleotides have shown evidence of immune modulation: intranasal administration of house dust mite (HDM) and cyclic GMP-AMP (cGAMP) can promote TH2 responses (12). Moreover, it has been shown that ST reprograms epithelial signaling and that the SJFα inflammatory cytokines induced by LT in animals are damped in the presence of ST (3), interactions that could alter mucosal immune system function. Recent antibody analysis of sera from individuals challenged with ST-only ETEC TW10722 suggests that mucosal immune responses are induced in the near term, but the longevity of immune responses wanes over time (13). Also, during heterologous ETEC challenge studies, the majority of volunteers who developed anti-LT serological responses following LT+ ST+-ETEC B7A exposure were still susceptible to LT+-ETEC E2528-C1-mediated challenge (14), supporting the idea that ST hinders development of sustained anti-LT immune or anti-ETEC.

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