Secretin and Secretin Receptor Are Required for Inflammatory Cell‐Cell Communication via Extracellular Vesicles Between Cholangiocytes Treated with Lipopolysaccharide
Authors: Sato Keisaku , Meng Fanyin , Venter Julie , Francis Heather , Glaser Shannon , Alpini Gianfranco 
Published: November 2023

BackgroundSecretin (SCT), 27 amino acids hormone, binds to secretin receptor (SR) and activates the cAMP‐dependent pathway during biliary damage leading to cholangiocyte proliferation. Secretin stimulation also increases exocytosis in cholangiocytes. Cholangiocyte proliferation is controlled by biliary extracellular vesicles (EVs), exosomes and microvesicles, which are released by exocytosis from cells. Cholangiocytes are heterogeneous and large but not small cholangiocytes express SR and perform ductular secretion. The cell‐cell communication between cholangiocytes and roles of cholangiocyte heterogeneity as well as the SCT/SR axis in EV secretion and functions are largely unknown. We identify cell‐cell communication between small and/or large cholangiocytes and determine the functional roles of SCT and SR in bacteria‐induced inflammatory cell‐cell communication.MethodsImmortalized normal mouse small and large cholangiocytes were cultured in EV‐depleted media. SCT and SR were stably knocked down by shRNA transfection in large cholangiocytes. Cells were stimulated with lipopolysaccharide (LPS, 50 ng/mL) or vehicle (PBS) for 72 hr. EVs were isolated from collected culture media by ultracentrifugation. After washes and another ultracentrifugation, EVs were resuspended in PBS. Small or large cholangiocytes were incubated with EVs isolated from small or large cells for 48 hr. Large cholangiocytes were also incubated with EVs isolated from negative control or gene‐knocked down large cholangiocytes. Then, total RNAs were harvested and RT‐qPCR was performed. The expression levels of proinflammatory cytokines (IL‐1β, IL‐6, TNFα and CCL2) were analyzed.ResultsLarge cholangiocytes secreted more EVs than small, and LPS stimulation increased EV secretion from large cholangiocytes but not from small cholangiocytes. LPS‐derived large EVs induced production of proinflammatory cytokines in other large cholangiocytes, but not in small cholangiocytes. LPS‐derived small EVs did not enhance cytokine production in small nor large cholangiocytes. Large cholangiocytes with knocked down SCT or SR secrete significantly reduced EVs compared with control large cholangiocytes. EVs isolated from SCT or SR‐knocked down cells treated with LPS did not induce elevated cytokine production in control large cholangiocytes.ConclusionThese novel findings show that cholangiocytes (following stimulation with LPS) activate inflammatory responses in other cholangiocytes via secretion of EVs. This cell‐cell communication occurs only between large cholangiocytes and small cholangiocytes do not participate in this event. Both SCT and SR are required for this inflammatory communication as well as effective EV secretion during LPS stimulation. This cell‐cell communication between cholangiocytes may be important to cause damage in other cholangiocytes, and EVs may be a novel therapeutic tool to regulate inflammatory reactions during biliary damage.Support or Funding InformationSupported in part by the Dr. Nicholas C. Hightower Centennial Chair of Gastroenterology from Scott & White, a VA Research Career Scientist Award, a VA Merit Award (5I01BX000574 to G.A.), US Department of Veterans Affairs Biomedical Laboratory Research VA Merit Awards (1I01BX001724 to F.M., 5I01BX002192 to S.G., and 1I01BX003031 to H.F.), NIH grants DK058411, DK076898, DK107310, and DK062975 (G.A., F.M., and S.G.), and NIH RO1 (1R01DK108959‐01 to H.F.). This material is the result of work supported by resources at the Central Texas Veterans Health Care System.