Improved survival ratios correlate with myeloid dendritic cell restoration in acute-on-chronic liver failure patients receiving methylprednisolone therapy
Cellular & Molecular Immunology
Improved survival ratios correlate with myeloid dendritic cell restoration in acute-on-chronic liver failure patients receiving methylprednisolone therapy
Acute-on-chronic liver failure (ACLF) is a severe life-threatening complication. Liver transplantation is the only available therapeutic option; however, several limitations have restricted its use in patients. The use of corticosteroids as an optional therapy for ACLF has received a great deal of interest. The rationale behind its use is the possible role of the immune system in initiating and perpetuating hepatic damage. In order to assess the relationship between myeloid dendritic cells (mDCs) and the efficacy of methylprednisolone (MP) treatment for hepatitis B virus (HBV)-associated ACLF patients, we recruited 30 HBV-associated ACLF patients who had received MP treatment at 10-day intervals; 26 patients received conservative medical (CM) management as a control. The functionality of DC subsets was lower in these ACLF patients compared with healthy subjects. In addition, compared with survivors, dead/transplanted patients had lower functional mDC in both groups. Furthermore, a decreased numbers of mDC at baseline was associated with high mortality of ACLF patients. Importantly, MP treatment resulted in a significant decrease in 28-day mortality, and all MP patients exhibited an initial rapid decrease in circulating mDC numbers within 10 days of MP treatment. Subsequently, MP survivors displayed a continuous increase in mDC numbers accompanied by a decrease in total bilirubin levels by more than 30%. However, MP dead/ transplanted patients lacked these sequential responses compared with survivors. This evidence suggests strongly that the higher mDC numbers at baseline and the recovery of mDC number at the end of treatment may represent a prognostic marker for favorable response to corticosteroid treatment in ACLF patients. Cellular & Molecular Immunology (2012) 9, 417-422; doi:10.1038/cmi.2011.51; published online 9 January 2012
acute-on-chronic liver failure; methylprednisolone; myeloid dendritic cells; plasmacytoid dendritic cells
Acute-on-chronic liver failure (ACLF) refers to acute hepatic insult
characterized by jaundice and coagulopathy, complicated within 4
weeks by ascites and/or encephalopathy in patients who had been
diagnosed previously or undiagnosed with chronic liver disease.1 In
some regions that have high hepatitis B virus (HBV) prevalence, the
term HBV-associated acute-on-chronic liver failure has been
introduced to describe acute aggravation of chronic hepatitis B liver failure,2
and this description constitutes about 70% of all the ACLF cases.1,2
HBV-ACLF is associated with high mortality and is a severe
lifethreatening condition. Plasma exchange and hemodiafiltration have
been used as artificial liver support systems for these patients, but these
treatments are only partially effective. Liver transplantation is
considered to be the standard treatment for these patients,3 but several
limitations such as a limited numbers of donors, long waiting lists, high
cost and multiple complications (e.g., rejection, problems associated
with the long-term use of immunosuppressants, and perioperative
morbidity and mortality) have restricted the use of liver
transplantation in many ACLF patients.4,5 Therefore, new strategies to delay or
prevent disease progression of liver failure are required urgently.
There is paramount evidence that immune dysregulation occurs in
HBV-ACLF patients.6?11 These changes include exacerbated innate
immunologic responses, such as the activation of
monocytes/macrophages and lymphocytes and cytokine release from these
inflammatory cells. Clinical findings and experimental rodent models suggest
that corticosteroids can suppress the effects of these activated
inflammatory cells.12,13 However, only some of the HBV-ACLF patients
treated with corticosteroids were cured without liver transplantation.
As such, the identification of immune parameters as indicators for the
efficacy of corticosteroid treatment is urgently needed.
Dendritic cells, as important professional antigen-presenting cells,
participate actively in driving the progression of ACLF.7,8,14 Two distinct
circulating dendritic cell (DC) subsets, myeloid and plasmacytoid DCs
(mDCs and pDCs), have been identified in humans based on phenotypic
and functional characterizations.15,16 In general, human mDCs are well
equipped to induce type 1 T helper cell polarization via the production
of interleukin-12 (IL-12) or tumor-necrosis factor-a, which plays a
crucial role in the development of liver damage. pDCs are characterized by
their distinct capacity to produce large amounts of type-1 interferon
(IFN), a key effector for inflammatory activation. Recent studies have
demonstrated the functional impairment of mDCs and pDCs in patients
with chronic HBV infection.17?19 Notably, these dendritic cells
accumulate mostly in the liver of ACLF patients;7,8,14 however, any
correlation between the DCs and corticosteroid efficacy is unknown.
In our present study, we examine the efficacy of methylprednisolone
(MP) treatment in 30 HBV-associated ACLF patients compared with
26 matched conservative medical managed ACLF patients. We found
that the higher mDC numbers at baseline or the recovery of mDC
numbers at the end of treatment may represent a prognostic marker
for favorable response to corticosteroid treatment in ACLF patients.
MATERIALS AND METHODS
Thirty patients with HBV-related ACLF received MP treatment, 26
patients who received conservative medical (CM) management and
30 healthy controls (HCs) were enrolled in this study. The project was
approved by the Ethics Committee of Beijing You An Hospital and
Beijing 302 Hospital; all patients signed a written informed consent
form in accordance with the Institutional Review Board guidelines for
the protection of human subjects. All HBV-related ACLF patients had
a history of chronic hepatitis or liver cirrhosis with serum HBsAg1 .6
months and had undergone the conventional treatment in this 4-week
study. Briefly, conservative medical management includes nutritional
support, hepatoprotective drugs, antiviral therapy, prevention and
control complications, as well as liver support device usage. In the MP
treatment group, patient?s treatment further consisted of 1mg/kg/day
(average: 80 mg/day) MP intravenously guttae for the first 3 days,
thereafter 0.75 mg/kg/day (average: 60 mg/day) intravenously guttae
continuously for the second 3 days and followed by 0.5 mg/kg/day
(average: 40 mg/day) intravenously guttae until the end of the third
3-day period (Figure 1). The baseline clinical parameters were matched
in the treatment and control groups (Table 1). Both groups of patients
were divided subsequently into non-survivor (NS) patients (who died
or received a liver graft) and survivors (who survived or whose total
bilirubin level decreased by more than 30% at the end of study).
Exclusion criteria were: history of variceal bleeding, recent
infection, bleeding tendency and/or ascites ultrafiltration and/or dialysis
during the last 2 months before enrolment; the presence of severe
renal, respiratory or cardiac disease or any detectable tumor by
ultrasonography, computed tomography or magnetic resonance
imaging; evidence of extrahepatic biliary diseases (e.g., presence of
primary sclerosing cholangitis or dilated common bile duct by
ultrasonography); detection of hepatic, portal or splenic vein
thromboses by Doppler ultrasonography; active substance abuse;
lack of a supportive family; and unwillingness to sign the informed
Flow cytometric analysis and cytokine release
The peripheral DC subset frequency was analyzed using
protocols described previously by our team and with minor
modifications.19,20 In brief, freshly isolated peripheral blood mononuclear
cells (PBMCs) were incubated with anti-lineage-1-FITC,
antiHLA-DR-PerCP and anti-CD11c-APC (BD Pharmingen, San Jose,
CA, USA) or anti-CD123-APC (Miltenyi Biotec, Bergish Gladbach,
Germany). The cells were fixed and analyzed by FACSCalibur and
CellQuest software (BD Biosciences, San Jose, CA, USA). At least 200
000 cells per run were acquired. The absolute numbers of circulating
DC subsets were calculated as per the formula: i.e., the counts of DCs
(cells/ml)?(the DC frequency of PBMCs)3(lymphocyte cell count/
ml1monocyte cell count/ml). Lymphocyte and monocyte counts
were determined by an automated differential blood counter. DC
subset function was evaluated by cytokine production. Freshly
prepared PBMCs were stimulated in 24-well plates with either
medium alone, 50 mg/ml poly(I:C) (Amersham Biosciences,
Piscataway, NJ, USA) or 6 mg/ml CpG 2216 (Sangon, Shanghai,
China) for 24 h at 37 uC. Then, supernatants were harvested and
tested in duplicate for IFN-a or IL-12 by use of a commercial
enzyme-linked immunosorbent assay kit according to the
manufacturer?s instructions (Biosource, Nivelles, Belgium). Sensitivity of the
assays was 10 pg/ml.
All data were analyzed using SPSS 13.0 for Windows software (SPSS
Inc., Chicago, IL, USA). Multiple comparisons were made among the
different groups using the Kruskal?Wallis test for non-parametric
data. Comparisons between various individuals were performed using
the Mann?Whitney U test; comparisons between parameters in the
same individual were performed using the Wilcoxon matched-pairs
ttest. Follow-up time was calculated as the interval between the date of
treatment and last follow-up or death. Survival rates were analyzed
with the Kaplan?Meier method using the log-rank test to assess the
difference between survival curves. For all tests, two-sided P values of
,0.05 were considered to be statistically significant.
Decreased numbers of functional DC subset cells in ACLF patients
The frequency of DC subset cells in the blood between ACLF patients
who received MP treatment or CM management was compared at
baseline; two cell populations of mDCs defined as
Lin-12HLADR1CD11c1 and pDCs with the Lin-12HLA-DR1CD1231
phenotype were monitored using flow cytometric analysis. In this study,
mDCs identified by CD11c expression may, in part, include the
CD11c1CD161 DC subset. As shown in Figure 2a and b, the numbers
of mDCs and pDCs were significantly lower (P,0.05) in the ACLF
group compared with that in the HC group: i.e., mDCs: 5.98 cells/ml
(range: 0.51?16.46 cells/ml) for the MP group; 5.66 cells/ml (range:
1.09?13.82 cells/ml) for the CM group vs. 19.49 cells/ml (range:
12.36?28.82 cells/ml) for HCs; and pDCs: 2.09 cells/ml (range: 0.02?
7.92 cells/ml) for the MP group; 1.84 cells/ml (range: 0.19?9.50 cells/ml)
for the CM group vs. 8.91 cells/ml (range: 6.36?17.49 cells/ml) for HCs.
No significant difference in DC subset counts was found between the
MP and CM groups.
We examined the profiles of cytokine released by DC subsets in
ACLF patients and in HCs. As shown in Figure 2c, a significant
reduction of poly(I:C)-induced IL-12 production was found in the MP
group and CM group compared with the HC group (313.9 pg/ml
(range: 106.2?625.8 pg/ml)) (P,0.05), however, there was no
significant difference in IL-12 production between the MP group (67.3 pg/
ml (range: 27.8?230.4 pg/ml)) and the CM group (65.4 pg/ml (range:
A significant reduction in CpG-induced IFN-a production by pDCs
was found in the MP group (130.9 pg/ml (range: 62.9?310.3 pg/ml)),
and in the CM group (116.4 pg/ml (range: 60.9?197.0 pg/ml)), when
compared with HCs (712.9 pg/ml (range: 138.5?1317.4 pg/ml))
(Figure 2d). However, there was no significant difference between
the MP group and the CM group.
These data indicate that a significant reduction in the numbers of
circulating DC subset cells occurs in ACLF patients.
ACLF outcome subgroup analysis
We further divided the ACLF patient group into either survivors who
survived without receiving a liver graft or NS patients who underwent
transplantation or died without transplantation. As shown in Figure 3a,
the numbers of mDCs in NS patients at baseline were significantly
lower than the numbers in survivors in both the MP group and the
CM group (MP group: 7.21 cells/ml (range: 1.36?16.46 cells/ml) for
survivor vs. 3.53 cells/ml (range: 0.51?9.06 cells/ml) for NS, P50.012;
CM group: 7.30 cells/ml (range: 1.19?13.82 cells/ml) for survivor vs.
3.42 cells/ml (range: 1.09?6.60 cells/ml) for NS, P50.005). In addition,
as shown in Figure 3b, a significant reduction in poly(I:C)-induced
IL12 production was found in the NS group compared with survivors
(MP group: 76.6 pg/ml (range: 39.5?230.4 pg/ml)) for survivors vs.
48.7 pg/ml (range: 27.8?89.4 pg/ml), P50.017; CM group: 75.8 pg/ml
(range: 37.7?148.0 pg/ml)) for survivors vs. 51.3 pg/ml (range: 22.9?
97.7 pg/ml), P50.047). Interestingly, the decreased pDC numbers were
found only in NS patients in the MP group (2.67 cells/ml (range: 0.02?
7.92 cells/ml) for survivors vs. 0.96 cells/ml (range: 0.15?3.36 cells/ml) for
NS, P50.039, Figure 3c). Furthermore, there was no significant
difference in CpG-induced IFN-a production by pDCs between survivors
and NS patients (Figure 3d).
Decreased circulating mDCs at baseline predicts poor survival of
To investigate the association of mDCs with the survival of ACLF
patients, ACLF patients in MP group and CM group were divided into
two groups (high mDCs and low mDCs group) by the median value of
circulating mDC frequency, respectively. The results showed that the
low mDC group had significantly poorer survival rates compared with
the high mDC group (P,0.001) (Figure 4a for MP group and
Figure 4b for CM group).
Improved survival ratios correlate with mDC restoration in
MP-treated ACLF patients
We monitored further the effect of MP treatment on the survival ratios
of enrolled patients in this study. It was found that the survival ratios
were increased significantly in the MP group compared with that in
CM group at the end of 28-day follow-up period (log-rank test with
P,0.001; Figure 5). These data suggest that MP therapy can improve
the survival ratios in these ACLF patients.
We further found that MP patients exhibited an initially rapid
decrease in circulating mDC numbers within 10 days of MP treatment.
Subsequently, MP survivors displayed a continuous increase in mDC
numbers accompanied by a total bilirubin level decrease of more than
30% (Figure 6a). However, MP dead/transplanted patients lacked
these sequential responses compared with survivors. mDCs numbers
remained persistently low in this cohort of patients (Figure 6b). There
was no microbiological evidence of sepsis during the study period.
These data suggest that the recovery of mDC numbers at the end of
treatment may represent a prognostic marker for favorable response to
corticosteroid treatment in ACLF patients.
This study characterized circulating DC subsets and revealed their
correlation with the efficacy in ACLF patient treatment with MP. We found
that decreased mDC numbers at baseline were associated with a high
mortality in ACLF patients. Furthermore, the survivors on MP therapy
exhibited an initially rapid decrease and then recovery of circulating
mDC numbers, while NS lacked this alteration. In addition to an
enhanced liver function, improved survival ratios were also observed
in these MP-treated ACLF patients during the clinical follow-up.
The development of ACLF involves dysregulation of both the innate
and adaptive immune systems. Many different cell types, including T
lymphocytes, monocytes and DCs, are primed in ACLF, and these are
believed to play a pivotal role in the pathogenesis of this disease.6?11
Corticosteroids have been used for the treatment of severe acute
hepatic failure by either oral or intravenous administration. Mechanically,
corticosteroids can suppress the effects of activated macrophages, DC
subsets and other inflammatory cells,10?11 which indicates that there is
a reciprocal effect on the efficacy of therapy. However, this therapy
has not been shown to have a satisfactory clinical effect and
has not been established definitively.12,13 As such, we speculate that
finding immune markers as a surrogate for therapeutic efficacy would
define the beneficial effects of this therapy. The decrease in DC
numbers in peripheral blood may be an important phenomenon of ACLF
pathogenesis. Previous data from our laboratory have indicated that
both mDCs and pDCs infiltrate extensively the liver of ACLF patients
and have expressed mature phenotypes.8,14 Therefore, this DC
migration from the blood to the liver may lead to the decreased DC numbers
found in the blood, while infiltrated DCs in liver may be involved in
the pathogenesis of ACLF. This study found that the decreased
numbers of mDC at baseline was associated with a high mortality of ACLF
patients and further indicated that the mDC numbers of the host may
determine the outcome of ACLF patients. Importantly, within 10 days
of MP therapy, circulating mDC numbers initially rapidly decreased,
possibly indicating the apoptosis of circulating mDCs. These data are
in agreement with previous studies that showed that glucocorticoid
can induce the apoptosis of DCs.21 The sequential responses were
uniquely found in survivors but not in NS patients because mDC
recovery occurred drastically and almost simultaneously with
improved liver function. Although our data suggest that a strong
mDC recovery may be an important factor for favorable responses
to MP treatment, it was rational to speculate that the rebound of
circulating mDC numbers may not simply be a direction result of
the redistribution of mDCs from the liver, where they function as
effectors of ACLF pathogenesis, to the blood. The altered hepatic
microenvironment or improved liver function or the recruitment of
Monocyte counts (/ml)
Lymphocyte counts (/ml)
Abbreviations: ACLF, acute-on-chronic liver failure; ALB, albumin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; PT, prothrombin time;
PTA, prothrombin activity; TBIL: total bilirubin.
Values are expressed as mean6standard deviation (s.d.) or median (average).
mDC or mDCs generation at bone marrow may be one of the factors
which account for the rebound of mDCs.
Notably, the present study was conducted on HBV-associated
ACLF patients relative to acute hepatic failure.12,13,22 In addition,
the control and treatment groups were homogeneous in the etiology
of liver disease (all patients had chronic HBV infection related liver
disease) and were comparable in terms of their baseline clinical
characteristics (Table 1). Therefore, this design, at least to some extent,
provides more convincing data related to the efficacy of MP in the
treatment of ACLF patients. However, there are still several limitations
existing in this study. Firstly, we did not randomize the patients.
Secondly, we did not document the mDC alterations in livers of the
studied patients because liver biopsy carries a high risk in ACLF
patients. However, the present study also highlights mDC numbers
that should be considered as an indicator for ACLF patients who are
capable of responding to MP treatment.
In conclusion, we evaluated the efficacy of MP-treated ACLF patient
with altered numbers of mDCs. We found that there was a potential
role for mDC numbers in the prediction of prognosis in these patients.
Therefore, these findings will help to provide patients with better
COMPETING FINANCIAL INTERESTS
The authors have no financial conflict of interest.
This study is supported by the National Natural Science Foundation of
China (Nos. 30801039 and 81072424) and the major projects of viral
hepatitis from Beijing Municipal Science and Technology Commission
(No. H020920020890). We thank the volunteers who generously participated
in this study.
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