Pancreata were collected from all mice and analyzed by histology and quantitative reverse transcription polymerase chain reaction. benzo[a]pyrene) or antagonists and analyzed by circulation cytometry. Mice were given intraperitoneal injections of caerulein or saline, with or without lipopolysaccharide, to induce CP. Some mice were given intraperitoneal injections of AhR agonists at the start of caerulein injection, with or without an antibody against IL22 (anti-IL22) starting 2 weeks after the first caerulein injection, or recombinant mouse IL22 or vehicle (control) intraperitoneally 4 weeks after the first caerulein injection. Mice were exposed to normal air or cigarette smoke for 6 h/d for 7 weeks and expression of AhR gene targets was measured. Pancreata were collected from all mice and analyzed by histology and quantitative reverse transcription polymerase chain reaction. Pancreatic stellate cells and T cells were isolated and analyzed using immunoblot, immunofluorescence, circulation cytometry, and enzyme-linked immunosorbent analyses. RESULTS Mice given AhR agonists developed more severe pancreatic fibrosis (based on decreased pancreas size, histology, and increased expression of fibrosis-associated genes) than mice not given agonists after caerulein injection. In mice given saline instead of caerulein, AhR ligands did not induce fibrosis. Pancreatic T cells from mice given AhR agonists and caerulein were activated and expressed IL22, but not IL17 or interferon gamma. Human T cells exposed to AhR agonists up-regulated expression of IL22. In mice given anti-IL22, pancreatic fibrosis did not progress, whereas mice given Dihydrokaempferol recombinant IL22 experienced a smaller pancreas and increased fibrosis. Pancreatic stellate cells isolated from mouse and human pancreata expressed the IL22 receptor IL22RA1. Incubation of the pancreatic stellate cells with IL22 induced their expression of the extracellular matrix genes fibronectin 1 and collagen type I and displayed as fold induction over untreated controls unless normally stated. Cell Preparation Pancreatic leukocytes were isolated using collagenase digestion method as explained previously for circulation cytometry analysis.13,16 PSCs from CP mice or human surgical specimens were isolated by outgrowth method as explained.13 Murine PSCs were cultured in Dulbeccos modified Eagle medium/F12 (1:1) containing 10% fetal bovine serum and were ready for use after the second passage. In Vitro T-Cell Differentiation Human peripheral blood mononuclear cells were isolated from buffy coat (blood lender) or blood from volunteer healthy smokers or nonsmokers by Ficoll-Hypaque density gradient centrifugation, and then na?ve CD4+ (from buffy coat) or total CD4+ (from smoker or nonsmoker healthy volunteer blood) T cells were purified with magnetic beads (Miltenyi Biotec) and cultured at 105 cells per well in 96-well round bottom plates. Enriched T cells were then stimulated for 5 days using plate-bound antibody to CD3 (1 (BioLegend), AF700-interferon gamma (BD, San Jose, CA), and matched isotype controls were utilized for intracellular staining. For human T-cell intracellular staining, phycoerythrin-IL22, allophycocyanin-IL17test was used to determine statistical significance between 2 groups and value <.05 was considered significant. Values are expressed as mean SEM or mean SD (Prism 5, GraphPad Software, San Diego, CA). Unless indicated, results are from at least 2C3 impartial experiments with 4 mice per group. Results Aryl Hydrocarbon Receptor Activation Worsens Fibrosis in Chronic Pancreatitis Cigarette smoking is an impartial risk factor for accelerating CP,6,9 however, the mechanism remains elusive. Cigarette smoke contains AhR agonists, such as dioxin and BaP10,11; in addition, cigarette smoke was found to have an unexpectedly high dioxin-like potential that triggers AhR activation.12 Therefore, we sought to investigate the role of cigarette smoke AhR ligands on immune activation and on the pathogenesis of CP. The well-characterized potent AhR agonist TCDD was administrated to mice undergoing caerulein-induced CP. Compared with the vehicle group, TCDD treatment worsened fibrosis in CP, as shown by Dihydrokaempferol decreased pancreas size, histology, and increased fibrosis-associated gene expression, such as ((fibronectin 1), (Collagen 1A1), and (transforming growth factorC[TGF(mean SEM, one-way ANOVA, Tukeys post-hoc test). (((collagen1A1), (fibronectin), (TGF(IL22) gene expression in the pancreas from VE- or TCDD-treated WT mice (mean SEM, unpaired 2-tailed Student test). (test). ((imply SEM). (expression in the pancreas from VE- or BaP-treated mice. Bar graphs show mean SEM (n = 9 Dihydrokaempferol per group, unpaired 2-tailed Student test). Fibrosis-promoting effects of AhR ligand TCDD are also seen in another CP model induced via chronic administration of caerulein and LPS Rabbit Polyclonal to CLNS1A (Supplementary Physique 3). Due to ubiquitous AhR expression pattern, we Dihydrokaempferol set up BM chimera studies to determine the contribution of hematopoietic cell AhR activation in the observed fibrosis progression. WT recipients were engrafted with WT or AhRd BM Dihydrokaempferol cells to.