Myeloid dendritic cells maintain Th1 7 lymphocyte responses during the obstructive phase of experimental biliary atresia
Celine S. Lages1, Julia Simmons1, Claire Chougnet3, Kumar S. Shanmukhappa2, Avery Maddox1, Betsy DiPasquale2, Alexander G. Miethke1;
1Pediatric Gastroenterology, Hepatology & Nutrition, Cincinnati Children's Hospital Medical Center, Cincinnati, OH; 2Pediatric Pathology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH; 3Immunology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH
Plasmacytoid dendritic cells (DC) were reported to drive innate injury to biliary epithelium during the early phase of biliary atresia (BA). We hypothesize that myeloid (m)DC coordinate T-cell injury during the obstructive phase of the disease. Methods: Transgenic CD1 1c-DTR GFP (DC reporter) mice were used to study DC in the pathogenesis of rhesus rotavirus induced BA. Immune responses were determined by cytometry, real time RT PCR and Luminex-based quantification of plasma cytokines. Results: Following injection on day 1 of life, the frequency of total DC increased by 6fold in the liver (1.2 % of CD1 1c+/CD3-7AAD- vs 0.2% in saline control, p<0.001) at 12 days post infection (dpi), with particular expansion of the CD11b+ mDC subset (0.4% vs 0.05% in saline, p<0.01). Since CD1 03+ mDC were reported to link innate with adaptive immune responses at inflammatory sites, we compared their frequency in liver and primary lymphoid tissue and found 2-fold increase of CD1 03+ cells in the mDC subset in the liver (1 6.5% of CD103+/mDC vs 8.5% in spleen, p=0.03). Immunohisto-chemistry (IHC) revealed accumulation of GFP+ immune cells in portal tracts, especially around bile duct profiles, at 12dpi. Accumulation of mDC was accompanied by intrahepatic expansion of CD4+ T cells by 4.3fold compared with non-infected controls (p<0.01). Hepatic mRNA concentration for IL17A was increased by 9.4fold between 8 and 12dpi after viral challenge. To investigate the effects of DC on CD4+ cells and Th1 7 responses in the liver after viral challenge, 0.01ng/gram bw of diphtheria toxin (DT) was injected every 48 hours beginning at 3dpi. The number of CD1 1c+ DC was significantly reduced in DT compared with vehicle treated mice at 8dpi (2.5x10^4/100mg tissue vs 5.0 in vehicle group, p<0.01). Anti-GFP-IHC demonstrated near absence of CD1 1 c+ immune cells from portal tracts. DC-depletion reduced CD4+ T cell content in the liver (5.0 vs 14.2x1 0^4/l 00mg tissue in vehicle; p=0.02) and their activation (24.1% vs 39.1% of CD69/CD4 in vehicle, p=0.03) at 12dpi. Hepatic mRNA expression of IL1 7A was down-regulated by 2.3fold (p=0.05), and plasma Th1 7-associated cytokine levels were reduced in DT treated mice compared with controls at 12dpi (IL-17F: 147.2 pg/ml vs 367.5, p=0.04; IL6: 21.3 and 34.5pg/mL, p=0.07, in DT vs vehicle treated mice, respectively). Importantly, diminished hepatic CD4+ cells and Th1 7 responses were associated with reduced plasma total bilirubin levels in DC-depleted mice at 12dpi (6.1 vs 1 1.3mg/dL in control; p<0.01). Conclusions: Myeloid DC maintain hepatic CD4 and Th1 7 responses and regulate cholestatic liver injury during the obstructive phase of experimental BA.
The following people have nothing to disclose: Celine S. Lages, Julia Simmons, Claire Chougnet, Kumar S. Shanmukhappa, Avery Maddox, Betsy DiPasquale, Alexander G. Miethke