Figure 1. Map of HBV construct
The pAAV/HBV1.2 plasmid was constructed by L.R. Huang. An over-length HBV
whole genome DNA was cloned into a pAAV vector. The plasmid is able to express
HBV transcripts and proteins in mouse hepatocytes and to secrete HBsAg, HBeAg, and
HBcAg.
35
Figure 2. Flowchart of KC depletion experiment.
6-week-old (or 12-week-old) male C3H/HeN mice were divided into three groups
which were intravenously injected clodronate liposome, control liposome or PBS,
respetively, at D-2. 2 days later (D0), all mice were proceeded hydrodynamic injection
of 10μg pAAV/HBV1.2 resolved in 8% body weight of PBS. HBV persistent rate was
evaluated by tracking the level of HBsAg in serum every week.
36
37
Figure 3. The efficiency of KCs depletion by using clodronate liposome
(A) Young and (B) adult C3H/HeN mice were proceeded KCs depletion treatment by
using clodronate liposome. The efficiency of KCs depletion was evaluated by flow
cytometry 2 days after the intravenous injection of clodronate liposome. The
populations of liver macrophages from young / adult C3H/HeN mice were acquired and
showed that vast KCs were depleted. The population of infiltrating macrophages
(MACs) increased after the depletion of KCs. (C) (D) (E) (F) The statistics showed that
infiltrating macrophages of adult C3H/HeN mice increased after the depletion of KCs.
38
(*) P < 0.05, (**) P < 0.01, (***) P <0.001. (n = 3~5).
39
40
Figure 4. HBV persistent rate of young C3H/HeN mice was reduced after the depletion of KCs
(A) The body weight of young C3H/HeN mice. (B) Young male C3H/HeN mice were
divided into three groups, one experimental group treated clodronate liposome (CLD, n
41
= 9)20 and two control group which were PBS control (PBS, n = 8) and vehicle control
(VEH, n = 12). CLD group showed significant decrease of HBV persistent rate
compared with control groups at 20 weeks after HDI of pAAV/HBV1.2. (C) The body
weight of adult C3H/HeN mice. (D) Adult male C3H/HeN mice were divided into
experimental group treated clodronate liposome (CLD, n = 10) and vehicle control
group (VEH, n = 5). (*) P < 0.05, (**) P < 0.01, (***) P <0.001. The follow-up of serum
and body weight were assisted by Dr. Wu and Pei Hsuan.
42
43
44
Figure 5. HDI time course of young C3H/HeN mice
(A) Young C3H/HeN mice were sacrificed 18 hours, 2 days, 3 days or 9 days after HDI.
The KC-enriched NPCs were isolated and analyzed by flow cytometry. Liver
macrophages were divided into two populations by F4/80 and CD11b. (B) (C) (D) (E)
MACs became dominate population after HDI and then gradually decreased in all
groups. The populations of liver macrophages were restored to baseline at day 9. (F)
The levels of HBsAg in serum were detected at 18 hours, 2 days, 3days and 9 days after
HDI. (n = 3~5). The isolation of KCs was assisted by Dr. Wu and Pei Hsuan.
45
46
47
Figure 6. HDI time course of adult C3H/HeN mice
(A) Adult C3H/HeN mice were sacrificed 18 hours, 2 days, 3 days or 9 days after HDI.
The KC-enriched NPCs were isolated and analyzed by flow cytometry. Liver
macrophages were divided into two populations by F4/80 and CD11b. (B) (C) (D) (E)
MACs became dominate population after HDI and then gradually decreased in all
groups. KCs were dramatically reduced day 2 after transfected HBV. The populations of
liver macrophages were restored to baseline at day 9 in all groups. (F) The levels of
48
HBsAg in serum were detected at 18 hours, 2 days, 3days and 9 days after HDI. (*) P <
0.05, (**) P < 0.01, (***) P <0.001. (n = 3~5). The isolation of KCs was assisted by Dr.
Wu and Pei Hsuan.
49
Figure 7. Flowchart of antibiotic experiment
C3H/HeN mice were treated antibiotic cocktail (ABX) in drinking water from 5- to
12-week-old. Mice were transfected HBV by HDI 2 days after withdrawal of antibiotic
cocktail and sacrificed 3 days after HDI.
50
51
52
Figure 8. KCs of antibiotics-treated adult C3H/HeN mice decreased at day 3 after the exposure to HBV
(A) Antibiotic-treated adult C3H/HeN mice (ABX) were sacrificed at day 3 after HDI.
The KC-enriched NPCs were isolated and analyzed by flow cytometry. Liver
macrophages were divided into two populations by F4/80 and CD11b. (B) KC
population of ABX group 3 days after HDI of pAAV/HBV1.2 was significantly less
53
than the control groups which received HDI of PBS or empty vector. (C) The
population of KCs from ABX group was significantly less than adult and young C3H/HeN mice that didn’t received antibiotic treatment. (D) The population of
infiltrating macrophages from ABX group was significantly higher than adult and young C3H/HeN mice that didn’t received antibiotic treatment. (E) The populations of liver
macrophages was similar in the baseline of adult C3H/HeN mice with or without
antibiotic treatment. (F) The comparison of the HBsAg level between antibiotics-treated
C3H/HeN mice and adult C3H/HeN mice. (*) P < 0.05, (**) P < 0.01, (***) P <0.001.
(n = 5~7). The isolation of KCs was assisted by Dr. Wu and Pei Hsuan.
54
Figure 9. The loss of TLR4 signaling resulted in dramatic reduction of HBV persistent rate.
6-week-old C3H/HeN (n = 8) and C3H/HeJ (n = 17) mice were transfected HBV by
HDI. The HBV persistent rate was acquired by tracking HBsAg in serum every week.
HBsAg persistent rate was less than 10% at 9th week after HDI in C3H/HeJ mice,
whereas it was 100% in C3H/HeN mice. (*) P < 0.05, (**) P < 0.01, (***) P <0.001.
55
56
57
Figure 10. Kupffer cell population of young C3H/HeJ mice decreased after the transfection of HBV
(A) KC-enriched NPCs were isolated from 6-week-old naive C3H/HeN and C3H/HeJ
mice and analyzed by flow cytometry. (B) KC-enriched NPCs were isolated from
6-week-old C3H/HeN and C3H/HeJ mice 18 hours, 2 days and 3 days after transfection of
HBV and analyzed by flow cytometry. Infiltrating macrophages and KCs were divided
58
by CD11b and F4/80. (C) The comparison of infiltrating macrophage population
between among young C3H/HeN, adult C3H/HeN and young C3H/HeJ. (D) The
comparison of KC population between among young C3H/HeN, adult C3H/HeN and
young C3H/HeJ. (*) P < 0.05, (**) P < 0.01, (***) P <0.001. (n = 3~5). The isolation of
KCs was assisted by Dr. Wu and Pei Hsuan.
59
60
Figure 11. Gating strategy of liver macrophages
KC-enriched NPCs isolated from mouse liver were analyzed by flow cytometry. After
gating out the debris of NPCs on light scatter, we selected CD45+ cell and divided
NPCs into KCs and infiltrating macrophages by CD11b and F4/80. The CD11bhigh
F4/80low population is infiltrating macrophages (Mac) and CD11blow F4/80high
population is KCs.
61
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