| Dendritic cells (DCs) are potent antigen-presenting cells (APCs), which bridge innate and adaptive immune responses and shape the balance between induction of immunity and tolerance. As sentinels, DCs alert the immune system, and determine the quantity and quality of the emerging immune response. Persistence of hepatitis B virus (HBV) infection is associated with reduced anti-viral T cell responses. Impaired dendritic cell (DC) function was suggested as the cause of reduced T cell stimulation in chronic HBV carriers, however, its role in hepatitis B virus (HBV)-related chronic-on-acute liver failure is still largely unknown. To investigate the frequencies of circulating dendritic cell (DC) subsets and the function of monocyte-derived dendritic cells in patients with hepatitis B related acute-on-chronic liver failure, we assayed the circulating precursor DC subsets (including mDC and pDC) and the ability of MoDCs in patients at various stages of HBV infection.Peripheral blood was collected from hepatitis B related acute-on-chronic liver failure patients (ACLF, n=60) and chronic hepatitis B (CHB, n=40) as well as normal controls (NCs, n=20). Circulating mDC and pDC frequencies in peripheral blood mononuclear cells (PBMC) were analyzed by flow cytometric analysis. Purified monocytes were isolated by combination of Histopaque-1.077 and CD 14 Microbeads. Monocyte-derived dendritic cells (MoDCs) generated in vitro in the presence of interleukin (IL)-4 and granulocyte macrophage colony-stimulating factor upon activation by poly I:C. Costimulatory molecule expression and allostimulatory mixed lymphocyte reaction (AMLR)of MoDCs were detected in patients with hepatitis B 1. The peripheral mDC and pDC frequency in normal controls were 0.52%±0.04% and 0.31%±0.02%, respectively. In addition, the peripheral mDC and pDC frequency in patients with CHB were 0.53%±0.11% and 0.2%±0.03%, respectively. Those of ACLF were 0.18%±0.02% and 0.15%±0.02%. The number of circulating mDC decreased only in patients with hepatitis B related ACLF compared with that in normal controls. However, pDC numbers decreased in both CHB and ACLF patients. We observed a further decrease the pDC numbers in ACLF compared to CHB patients, however, but this difference did not achieve P< 0.05 statistical significance.2. After in vitro challenge with poly(I:C), the expression of NC-DC surface molecular such as MHC classⅡmolecules (HLA-DR), and the mature marker CD83, as well as costimulatory molecules CD86, CD80 were 302.1±5.6,205.1±13.7,346.7±14.6, and 38.8±1.0, respectively. But both groups showed low expressions of above surface molecular (272.2±3.38,173.0±6.4,315.5±7.93, and 30.6±0.57 in CHB group; 223.0±5.5,148.2±6.1,219.1±7.1, and 25.0±0.6 in ACLF group). MoDC from ACLF patients showed lower expression of costimulatory molecules CD80, CD86 and the mature marker CD83, as well as MHCⅡmolecule (HLA-DR) compared to CHB and NC group.3. Interestingly, MoDC impaired allostimulatory mixed lymphocyte reaction from ACLF patients compared with those in CHB patients and NCs.Our results suggest that patients with hepatitis B related ACLF have a significantly lower expression of surface markers and impaired AMLR of MoDC, as well as decreased number of circulating mDC and pDC, which may be partially related to HBV disease progression in these patients. Toll-like receptors (TLRs) are a class of proteins that play key roles in innate immunity through recognition of microbial components. TLR3 is expressed abundantly in dendritic cells, and is responsible for recognizing viral pathogens and inducing proinflammtory cytokine and interferon beta (IFN-β) production. Although TLR3 has been reported to be involved in several diseases caused by viral infections, its role in hepatitis B virus (HBV)-induced hepatitis is still largely unknown. This study aims to investigate the expression of TLR3 signaling in monocyte derived dendritic cells (MoDC) from ACLF, to assess the contribution of TLR3 in ACLF.Peripheral blood was collected from ACLF patients (n=60) and chronic hepatitis B (CHB, n=40) as well as normal controls (n=20). Purified monocytes were isolated by combination of Histopaque-1.077 and CD 14 Microbeads. Monocyte-derived dendritic cells (MoDCs) generated in vitro in the presence of interleukin (IL)-4 and granulocyte macrophage colony-stimulating factor upon activation by TLR3 ligand (poly I:C). TLR3 pathway genes of extracted RNA were detected using real-time PCR. The level of TLR3 protein was measured by flow cytometric analysis and western blotting. Supernatant fluids from the cultures were analyzed for levels of 6 different pro-inflammatory cytokines, interleukin (IL)-1β, IL-6, IL-8, IL-10, IL-12p70, and TNF-a by flow cytometric, and interferonβwas measured by ELISA.1. We found that expression of TLR3 mRNA was decreased significantly in monocyte-derived dendritic cells (MoDCs) from patients with CHB (41.5%) or ACLF (57.7%), compared with NCs. Particularly in ACLF patients, the TLR3 expression was further decreased under that in CHB patients (P<0.001). Data from western blotting and flow cytometry analysis further confirm the above observation at the protein level. In addition, the MFI of TLR3 was lower in HBV infection patients than NC group (P< 0.001), we observed a further decrease in TLR3 MFI in ACLF compared to CHB patients (P=0.006).2. Interestingly, ACLF-MoDC produced imbalanced relative levels of pro-inflammatory cytokines, particularly IL-6 and TNF-α, were higher than CHB patients and controls. While, aberrant amounts were found of IL-12p70, IL-10, IL-1β, and IL-8 on MoDCs in ACLF.3. The secretion of IFN-βfrom DC of control group, patients with CHB and ACLF were (156.10±6.98) pg/ml, (133.95±5.45)pg/ml, and (74.77±3.1) pg/ml, respectively. The secretion of IFN-βwas mirrored on the expression of TLR3. The secretion of IFN-βwas lower in patients with CHB and ACLF than NC group (P< 0.001). We observed a further decrease in IFN-P in ACLF compared to CHB patients (P< 0.001).4. Compared with surviving patients, TLR3 and IFN-βexpression was significantly lower in non-surviving ACLF patients, which strongly indicated a correlation between TLR3 signaling impairment in MoDCs and disease severity in ACLF patients. Further linear correlation analysis demonstrated significant correlations between expression of TLR3 signaling components (TLR3 and IFN-β) and disease severity markers (prothrombin activity and total bilirubin) for individual ACLF patients.The results suggest impaired TLR3 pathway signaling in MoDC from patients with ACLF, resulting in secretion of selective pro-inflammatory cytokines and IFN-βintegral to the inflammatory response that may be critical in the pathogenesis of ACLF.
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