Table 1.
Clinical characteristics of the populations enrolled in the study.
Figure 1.
Increased monocytes in peripheral blood and liver positively correlate with liver injury in IA patients.
(A) Absolute counts of the peripheral blood monocytes from IT (n = 32), IA (n = 78) patients and healthy controls (n = 38). Each dot represents the value from one individual. P values are shown. (B) The absolute numbers of peripheral blood monocytes was significantly correlated with serum ALT levels but not with HBV DNA. The solid line represents the linear growth trend and r, the correlative coefficient. P values are shown. (C) Representative immunohistochemical staining of CD68 in specimens from IA patients. An IA patient with HAI G2S2 scores has higher CD68+ cell density in liver, compared to that of a patient with HAI G1S1 scores. (D) Numbers of CD68+ cells in portal area are shown in IA patients with various degrees of liver HAI scores. Horizontal bars represent the median CD68+ cells. ALT, alanine aminotransferase; HAI, histological activity index; IT, Immune tolerant carriers; IA, immune activated patients.
Figure 2.
Peripheral CD16+ monocyte frequencies are significantly increased in IA patients.
(A) Monocyte subsets in whole blood were identified based on forward and side scatter (left panel) and CD14/CD16 expression patterns (right panel). Monocytes were further divided into the CD14highCD16− subset and a major CD16+subsets; the latter were further divided into CD14highCD16+ and CD14lowCD16+ subsets for analysis. (B) Representative dot plots of three monocyte subsets in IT, IA patients and healthy controls. Values in the quadrants indicate the proportions of monocyte subsets. (C) Individual values of the three monocyte subsets in IT, IA patients and healthy controls. P values are shown. Horizontal bars represent the median proportions of each monocyte subset. (D) Individual values of major CD16+ monocyte subsets in IT, IA patients and healthy controls. P values are shown. Horizontal bars represent the median proportions of the CD16+ monocyte subsets. IT, Immune tolerant carriers; IA, immune activated patients.
Figure 3.
Increased CD16+ monocytes in peripheral blood and liver positively correlate with liver injury in IA patients.
(A) The major CD16+ monocytes and CD14highCD16+ monocyte subsets were significantly correlelated with serum ALT levels, but not with HBV DNA. The solid line represents the linear growth trend and r, the correlative coefficient. P values are shown. (B) Representative dot plots of CD14 and CD16 staining in monocytes from peripheral blood and LILs isolated from the the same IA patient are shown. Values in the upper-left and upper-right quadrants represent the percentages of CD14highCD16− monocytes and CD16+ monocytes, respectively. (C) Individual percentages of hepatic and peripheral monocytes that express CD16 from the IA patients. Each dot represents one individual. P values are shown. (D) IA patients with higher HAI scores (G and S) have a higher percentage of monocytes expressing CD16 in their livers, compared to patients with lower HAI scores. P values are shown. ALT, alanine aminotransferase; HAI, histological activity index; LILs, intrahepatic leukocytes.
Figure 4.
Hyper-activated CD16+ monocytes preferentially produce Th17-related cytokines.
(A) Histogram plots of HLA-DR expression on monocytes and CD16+ monocytes from circulating monocytes (blood) and liver-infiltrating monocytes (liver) in a representative IA individual. (B) The MFI values of HLA-DR expressed on monocytes and CD16+ monocytes from CHB patients in each group. Each open dot represents one subject with lower HAI scores and each solid dot represents patients with higher HAI scores. (C) Representative histogram plots of TNF-α, IL-6, IL-1β, IL-12/23 p40 expression (red line histograms) or isotype control (green line histograms) on CD14+ CD16− monocytes and CD16+ monocytes in an IA subject. (D) Data shown are representative of ten independently performed experiments from IA subjects. (E) Representative expansion of Th17 cells with the cytokine cocktail (IL-6, IL-1β and IL-23) and TCR-stimulation. Values in the upper left quadrant indicate the percentages of CFSElow Th17 cells. (F) Individual values of the fold changes in the percentage of total and CFSElow Th17 cells. Horizontal bars indicate the median fold changes. (G) Representative expansion of Th17 cells with activated CD16+ monocytes in HC subjects (n = 4). Purified CD4+ T cells from HCs were cocultured with autologous CD16+ monocytes and anti-CD3/CD28 microbeads in the absence or presence of LPS. After 4 days, cells were stimulated for 6 h with PMA/ionomycin and Golgi-stop (during the last 5 h), and IL-17 expression and production were determined by ICC staining. Values in the upper quadrant represent the percentage of Th17 cells. MFI, mean fluorescence intensity; HAI, histological activity index; IA, immune activated patients; CFSE, Carboxyfluorescein succinimidyl ester.
Figure 5.
Increased neutrophils in liver in situ positively correlate with liver injury in IA patients.
(A) Absolute counts of peripheral blood neutrophils from IT (n = 32), IA (n = 78) patients and healthy controls (n = 38). Each dot represents one individual. P values are shown. (B) The absolute number of neutrophils significantly negatively correlated with the serum ALT levels, but not with HBV DNA. The solid line represents the linear growth trend and r, the correlative coefficient. P values are shown. (C) Immunohistochemical staining for MPO (specific for neutrophils) in the portal area in IA patients with HAI scores (G1S1 and G2S2) (400×). A G2S2 patient had a higher percentage of neutrophils in LILs compared to a G1S1 patient. Values in the upper right indicate the percentages of neutrophils in LILs. (D) IA patients with higher HAI scores (G and S) have a higher percentage of liver neutrophils in the portal area, compared to patients with lower HAI scores. Each dot represents one individual. P values are shown. Horizontal bars represent the median proportions of liver neutrophils. IT, Immune tolerant carriers; IA, immune activated patients. HAI, histological activity index; LILs, intrahepatic leukocytes.