BACKGROUND AND AIMS: Increased levels of chemokine interferon-gamma (IFN-γ)-inducible protein-10 (CXCL10), soluble CD163 (sCD163) and soluble CD14 (sCD14) have been reported in HCV infection. The aim of this study was to compare, sCD163 and sCD14 levels in HCV-infected patients undergoing direct acting antiviral (DAA)-containing regimens with or without interferon (IFN). METHODS: sCD163, sCD14 and CXCL10 were longitudinally measured by ELISA in 159 plasma samples from 25 HCV-infected patients undergoing IFN-based treatment plus telaprevir or boceprevir and 28 HCV infected subjects treated with DAA IFN-free regimens. Twenty-five healthy donors (HD) were included as controls. RESULTS: At baseline CXCL10, sCD163 and sCD14 levels were higher in HCV-infected patients than in HD. CXCL10 and sCD163 levels were significantly decreased in responder (R) patients who achieved sustained virological response (SVR), with both IFN-based and IFN-free regimens, while they were persistently elevated in non-responders (NR) patients who stopped IFN-based treatments because of failure or adverse events. Conversely, sCD14 levels were apparently unchanged during therapy, but at the end of treatment the levels reached normal ranges. Comparing the two regimens, the extent of CXCL10 reduction was more pronounced in patients undergoing DAA IFN-free therapies, whereas sCD163 and sCD14 reduction was similar in the two groups. Interestingly, only in IFN-based regimens baseline sCD163 levels were significantly higher in NR than in R patients, while in the IFN-free treatment group also patients with high sCD163 plasma levels obtained SVR. At the end of therapy, even if the biomarkers were largely decreased, their levels remained significantly higher compared to HD. Only in the early fibrosis stages, sCD163 values tended to normalize. CONCLUSIONS: These results indicate that IFN-free regimens including newer DAA induce an early and marked decrease in circulating inflammatory biomarkers. However, the full normalization of biomarkers was not obtained, especially in patients with advanced fibrosis, thus underlying the need for a treatment in the early stages of HCV infection.
BACKGROUND AND AIMS: Increased levels of chemokine interferon-gamma (IFN-γ)-inducible protein-10 (CXCL10), soluble CD163 (sCD163) and soluble CD14 (sCD14) have been reported in HCV infection. The aim of this study was to compare, sCD163 and sCD14 levels in HCV-infectedpatients undergoing direct acting antiviral (DAA)-containing regimens with or without interferon (IFN). METHODS: sCD163, sCD14 and CXCL10 were longitudinally measured by ELISA in 159 plasma samples from 25 HCV-infectedpatients undergoing IFN-based treatment plus telaprevir or boceprevir and 28 HCV infected subjects treated with DAAIFN-free regimens. Twenty-five healthy donors (HD) were included as controls. RESULTS: At baseline CXCL10, sCD163 and sCD14 levels were higher in HCV-infectedpatients than in HD. CXCL10 and sCD163 levels were significantly decreased in responder (R) patients who achieved sustained virological response (SVR), with both IFN-based and IFN-free regimens, while they were persistently elevated in non-responders (NR) patients who stopped IFN-based treatments because of failure or adverse events. Conversely, sCD14 levels were apparently unchanged during therapy, but at the end of treatment the levels reached normal ranges. Comparing the two regimens, the extent of CXCL10 reduction was more pronounced in patients undergoing DAAIFN-free therapies, whereas sCD163 and sCD14 reduction was similar in the two groups. Interestingly, only in IFN-based regimens baseline sCD163 levels were significantly higher in NR than in R patients, while in the IFN-free treatment group also patients with high sCD163 plasma levels obtained SVR. At the end of therapy, even if the biomarkers were largely decreased, their levels remained significantly higher compared to HD. Only in the early fibrosis stages, sCD163 values tended to normalize. CONCLUSIONS: These results indicate that IFN-free regimens including newer DAA induce an early and marked decrease in circulating inflammatory biomarkers. However, the full normalization of biomarkers was not obtained, especially in patients with advanced fibrosis, thus underlying the need for a treatment in the early stages of HCV infection.
Hepatitis C virus (HCV) is a major cause of liver disease worldwide, leading to
progressive fibrosis, potential development of cirrhosis and hepatocellular
carcinoma (HCC) [1]. For
years, the standard of care of HCV chronic infection has been the treatment with
interferon alpha (IFNα)-based regimens. The advances in therapy from the use of
standard IFNα monotherapy to pegylated IFNα (PEG-IFNα) in combination with ribavirin
(RBV) and, then, to first generation protease inhibitors telaprevir (TVR) and
boceprevir (BOC) resulted in improvements in the rates of sustained virological
response (SVR) [2].
Nevertheless, in the last years, a remarkable success in the management of HCV
infection was obtained with the introduction in clinical practice of several
all-oral IFN-free direct acting antiviral agents (DAAs). These new drug classes
improved SVR rate up to 95–100% of cases, increasing the possibility of HCV
clearance [3].Since IFN-free DAA regimens have specific steps of the virus life cycle as a target,
they help clarifying the interaction between HCV and the innate immune response,
regardless of the IFNα induced immune modulation [4]. It is well known that sustained
inflammation and fibrinogenesis represent the basis of liver damage during chronic
HCV infection [5]. This
process is associated with increased production of inflammatory chemokines and
mediators [6] and a permanent
activation of the innate immune system, including natural killer (NK) cells and
liver monocytes/macrophages, mostly Kupffer cells [7,8]. Various inflammatory and innate immune
activation biomarkers have been studied during chronic HCV infection and are
correlated with liver disease progression, as well as with the development of HCV
extrahepatic manifestations, such as cardio-cerebrovascular diseases [9-11]. The complex interactions between these
inflammatory biomarkers and the outcome of HCV infection have led to studies on the
effect of antiviral treatment for HCV. In the pre-DAAs IFN-based treatment era, the
analysis of circulating biomarkers improved the understanding of immune responses to
HCV infection and showed their usefulness in predicting therapeutic responses [12-14]. Among plasma biomarkers, we studied
chemokine interferon-γ-inducible protein-10 (CXCL10), soluble (s) CD163 and sCD14,
because they play an important role in the pathogenesis of HCV infection and have
been associated with hepatic inflammatory activity and fibrosis stage [15-17]. Nowadays, the evaluation of the effect of
newer IFN-free regimens on the temporal dynamics of inflammatory biomarkers
represents an area of active investigation.In the present study, we evaluated the effects of DAA-containing regimens with or
without IFN in a cohort of HCV infectedpatients, on dynamic changes in circulating
levels of the following biomarkers of innate inflammation and immune activation: i)
CXCL10, an inflammatory chemokine reflecting liver expression of interferon
stimulated genes; ii) sCD163, a marker of monocyte/macrophage activation; iii)
sCD14, a marker of immune activation and microbial translocation.
Patients and methods
Study population
The study was conducted in two out-patient clinics of a single referral center
(Sapienza University, Rome). The study population included 53 patients with
active HCV infection who were treated according to the current Italian national
guidelines for HCV treatment. Patients had no evidence of HIV or HBV infection
or decompensated liver disease. Based on treatment response, patients were
classified as responders (R) if they reached sustained virological response,
defined as undetectable HCV RNA in blood 12 weeks after the end of therapy
(SVR12). The subjects who stopped treatment because of virological failure
(NRvf) or side effects (NRse) were defined as non responders (NR). Twenty-five
healthy donors (48, 32–58 years; 42% male/female) were included as controls.The study was approved by the Ethics Committee of “Sapienza” University of Rome.
All subjects signed a written informed consent before enrolment in the study.
Data and plasma samples were collected respecting donor’s confidentiality and
privacy.
HCV-RNA and HCV genotype testing
Plasma HCV-RNA levels were determined by RealTime PCR Roche Cobas TaqMan. HCV
genotypes and subtypes 1a and 1b were determined by Abbott
RealTime HCV Genotype II.
Liver fibrosis assessment
Liver stiffness (measured in kPa) was determined by transient elastography with
the use of a Fibroscan machine (Echosens). Advanced fibrosis (F4) was defined as
a liver stiffness measure (LSM) greater than or equal to 14.5 kPa, severe
fibrosis (F3) was defined as a LSM greater than or equal to 10, mild or no
fibrosis (F0-F2) was defined as a LSM less than 10 kPa [18]. The biochemical index FIB-4 was also
used to assess liver fibrosis and calculated using Sterling's formula: age
[years] × AST [IU/L]/platelet count [expressed as platelets × 109/L]
× (ALT1/2 [IU/L]).
Samples handling
Venous blood samples were collected from each patient into EDTA and heparin
containing tubes (Becton–Dickinson Systems, San Jose, CA) and the cell-free
plasma was stored at -80°C until use. A total of 159 plasma samples were
collected from HCV-infectedpatients and 25 from healthy donors. The patients
were scheduled for plasma collection using the following timing: T0 = baseline
before therapy; T1 = after 4 weeks of therapy; T2 = 12 weeks after the end of
treatment for R patient or the time of failure (stopping rule) for NR
subjects.
Detection of soluble biomarkers
Commercially available ELISA kits were used for the quantitative detection of
plasma levels of CXCL10, sCD163 and sCD14 (Quantikine, R&D Systems,
Minneapolis, MN), according to the manufacturer’s instructions. To preserve the
linearity of the assays, samples containing high concentrations of CXCL10 or
sCD163 and sCD14 were diluted with an appropriate amount of calibrator diluent.
The reported minimum detectable doses of CXCL10, sCD163 and sCD14 were 1.67
pg/ml, 0.177 ng/ml and 0,125 ng/ml, respectively. All samples were tested in
duplicate.
Statistical analysis
All statistical analyses were performed using GraphPad Prism Software version 5
(Software MacKiev). Values are given as median and ranges. Non-parametric
Mann-Whitney test and non-parametric Kruskal-Wallis ANOVA with Dunn's post-test
were applied to compare the differences in values. Non-parametric Friedman test
with Dunn's multiple comparison test was applied to perform longitudinal
analyses. We assessed the correlations between inflammatory and clinical
parameters using Spearman’s correlation coefficient (r). All statistical
analyses were considered significant with p-values less than
<0.05.
Results
Study population characteristics
The clinical characteristics of the 53 HCV-infectedpatients receiving anti-HCV
therapy are shown in Table
1. Twenty-five subjects were treated with an IFN-based treatment
including PEG-IFNα/RBV plus first generation protease inhibitors (TVR or BOC),
whereas 28 subjects were treated with IFN-free DAA regimens with or without
RBV.
Results are expressed in median (range). For continuous variable
Mann-Whitney test and for variable dichotomous Chi-Square test were
performed. HCV-RNA: hepatitis C virus ribonucleic acid, TVR:
Telaprevir, BOC: Boceprevir, SOF: Sofosbuvir, SIM: Simeprevir, LED:
Ledipasvir, 3D: Ombitasvir, Paritaprevir, Dasabuvir, DAC:
Daclatasvir, ALT: alanine aminotransferase, AST: aspartate
aminotransferase, PLT: platelets, SVR: sustained virologic response,
NA: not applicable, NS: not significant.
Results are expressed in median (range). For continuous variable
Mann-Whitney test and for variable dichotomous Chi-Square test were
performed. HCV-RNA: hepatitis C virus ribonucleic acid, TVR:
Telaprevir, BOC: Boceprevir, SOF: Sofosbuvir, SIM: Simeprevir, LED:
Ledipasvir, 3D: Ombitasvir, Paritaprevir, Dasabuvir, DAC:
Daclatasvir, ALT: alanine aminotransferase, AST: aspartate
aminotransferase, PLT: platelets, SVR: sustained virologic response,
NA: not applicable, NS: not significant.In the IFN-based treatment group, 13 patients (52%) were responders and 12 (48%)
non-responders, of which 33% NRvf and 67% NRse. All patients treated with
IFN-free DAA regimens were responders, since they reached SVR12.
Soluble biomarkers plasma levels in treatment groups and healthy
controls
Plasma levels (median, ranges) of CXCL10 (412.8, 133.8–568.5), sCD163 (1572.6,
790.6–4119.7) and sCD14 (1732.1, 1007.5–3310) in HCV-infectedpatients at
baseline were significantly higher than in HD (81.3, 27.7–236.3; 457.6,
279.2–810.5; 1341.4, 917.4–2627.4, respectively) (Fig 1, Panel A).
Fig 1
Plasma levels of CXCL10, sCD163 and sCD14 in HCV-infected patients
and healthy controls.
Panel A: Box plots represent circulating levels of CXCL10 (n = 53),
sCD163 (n = 53) and sCD14 (n = 45) in HCV-infected patients at baseline
and in 25 HD. Statistical differences were assessed by Mann-Whitney and
p are indicated. Panel B: Box plot represents circulating levels of
CXCL10, sCD163 and sCD14 in HCV-infected patients according to fibrosis
stage at baseline. Horizontal bars represent the median values and
dashed lines indicate the median value of HD. Kruskal-Wallis ANOVA with
Dunn’s test were performed to assess statistical differences between the
different groups. HCV: hepatitis C virus; HD: healthy donors; F2-F0:
absent to mild liver fibrosis; F3: severe liver fibrosis; F4: advanced
liver fibrosis; NS: not significant.
Plasma levels of CXCL10, sCD163 and sCD14 in HCV-infected patients
and healthy controls.
Panel A: Box plots represent circulating levels of CXCL10 (n = 53),
sCD163 (n = 53) and sCD14 (n = 45) in HCV-infectedpatients at baseline
and in 25 HD. Statistical differences were assessed by Mann-Whitney and
p are indicated. Panel B: Box plot represents circulating levels of
CXCL10, sCD163 and sCD14 in HCV-infectedpatients according to fibrosis
stage at baseline. Horizontal bars represent the median values and
dashed lines indicate the median value of HD. Kruskal-Wallis ANOVA with
Dunn’s test were performed to assess statistical differences between the
different groups. HCV: hepatitis C virus; HD: healthy donors; F2-F0:
absent to mild liver fibrosis; F3: severe liver fibrosis; F4: advanced
liver fibrosis; NS: not significant.When we analyzed the levels of biomarkers according to fibrosis stage at
baseline, all groups showed higher levels of CXCL10 and sCD163 in comparison
with HD (p<0.001), while sCD14 levels were similar in subjects with F0-F2, F3
liver fibrosis and HD (p>0.05), and were increased only in cirrhotic subjects
(p = 0.03) (Fig 1, Panel
B).Patients with severe fibrosis (F4) had significantly higher sCD163 concentrations
(1785.7,1169.5–4119.7) compared to those with mild fibrosis (F0-F2) (1194,
790.6–2201.7) (p = 0.001), while no statistically significant differences for
CXCL10 and sCD14 levels were found. Moreover, CXCL10 levels positively
correlated with FIB-4 (r = 0.4; p = 0.003); sCD163 levels positively correlated
with FIB-4 (r = 0.5; p = 0.0004), hepatic stiffness (r = 0.5; p = 0.001), AST (r
= 0.4; p = 0.008) and negatively with viremia (r = -0.3; p = 0.03) and platelet
absolute counts (r = -0.3; p = 0.02).
Longitudinal changes in soluble biomarkers during treatment
CXCL10 levels
Changes in the levels of CXCL10 in IFN-based treatment group (R and NR) and
IFN-free DAA regimens are shown in Fig 2. At baseline in the IFN-based
treatment group, there was not a significant difference in CXCL10 levels
between NR (n = 12) and R subjects (n = 13) (p = 0.18). The median (ranges)
CXCL10 levels were 426.9 (142.3–568.5) in NR patients and 307.2
(159.9–495.4) in R patients. A significant decrease in CXCL10 concentrations
was found one month after initiation of therapy only in R patients and its
values remained low up to the SVR12 (T2) (p = 0.004). The median (ranges)
value of CXCL10 at baseline was 307.2 (159.9–495.4) and decreased to 249.41
(108.5–495.4) at T1 and 162.4 (115.2–457.8) at T2 (p = 0.004 for T2 versus
T0). Conversely, in NR patients, after an initial significant decrease at
T1, CXCL10 levels increased at the time of stopping rule. The median
(ranges) CXCL10 levels in NR patients were 426.9 (142.3–568.5) at T0, 229.8
(90.1–291.6) at T1 and 345 (164.3–1154.1) at the time of stopping rule (p =
0.02 for T1 versus T0).
Fig 2
Longitudinal changes in soluble biomarkers in HCV-infected
patients undergoing DAA containing regimen with or without
IFN.
Box plots represent circulating levels of CXCL10 (n = 53), sCD163 (n
= 53) and sCD14 (n = 45) in HCV-infected patients at T0, T1 and T2
in R patients during IFN-based treatment, at T0, T1 and T2 in NR
patients during IFN-based treatment and at T0, T1 and T2 during
IFN-free treatment. Box plots show 10th, 50th
(median), 90th, percentile and whiskers. Horizontal bars
represent the median values. Statistical analysis was performed
using Friedman test, p values indicate the no post-test. R:
responders; NR: non responders; T0: baseline before therapy; T1:
after 4 weeks of therapy; T2: 12 weeks after the end of treatment
for R patient or the time of failure (stopping rule) for NR
subjects.
Longitudinal changes in soluble biomarkers in HCV-infected
patients undergoing DAA containing regimen with or without
IFN.
Box plots represent circulating levels of CXCL10 (n = 53), sCD163 (n
= 53) and sCD14 (n = 45) in HCV-infectedpatients at T0, T1 and T2
in R patients during IFN-based treatment, at T0, T1 and T2 in NR
patients during IFN-based treatment and at T0, T1 and T2 during
IFN-free treatment. Box plots show 10th, 50th
(median), 90th, percentile and whiskers. Horizontal bars
represent the median values. Statistical analysis was performed
using Friedman test, p values indicate the no post-test. R:
responders; NR: non responders; T0: baseline before therapy; T1:
after 4 weeks of therapy; T2: 12 weeks after the end of treatment
for R patient or the time of failure (stopping rule) for NR
subjects.In the group of patients treated with IFN-free DAA regimens, there was a
marked decrease of CXCL10 plasma levels at T1 and its values remained low up
to SVR12 (T2) (p<0.0001). The median (ranges) CXCL10 levels were the
following: 451.8 (133.8–568.5) at T0, 123.2 (81.5–568.5) at T1, 137.4
(36–517.6) at T2 (p<0.0001 for both T1 and T2 versus T0).
sCD163 levels
The basal sCD163 levels in the IFN-based treatment group were significantly
higher in NR patients (median 1702.8, ranges 1139.8–2201.7) than in R
patients (1208.1, 790.6–1570.2) (p = 0.0005). During the IFN-based
treatment, sCD163 levels significantly decreased in R patients only at T2
(SVR12) (p<0.0001). The median (ranges) sCD163 levels were 1208.1
(790.6–1570.2) at T0, 1062.6 (755.1–1372.1) at T1 and decreased to 635.6
(462.3–1332.1) at T2 (p<0.0001 T2 vs T0). Conversely, in NR patients,
after an initial significant decrease at T1, sCD163 levels increased at the
time of stopping rule. The median (ranges) sCD163 levels in NR patients were
1702.8 (1139.8–2201.7) at T0, 1178.1 (1098.5–1591.4) at T1, 1546.5
(717.8–2201.7) at the time of stopping rule (p = 0.02 for T1 versus T0)
(Fig 2).In patients treated with IFN-free DAA regimens, sCD163 levels decreased after
one month of therapy and its values remained low up to SVR12 (T2) with only
a moderate increase (p = 0.0004). The median (ranges) sCD163 levels were the
following: 1905 (1124.1–4119.7) at T0, 1161.7 (617.2–3044) at T1, 1524.1
(563.2–4002.3) at T2 (p = 0.0004 for T1 versus T0) (Fig 2).
sCD14 levels
Unlike sCD163, there was not a significant difference in sCD14 levels between
NR and R subjects at baseline in the IFN-based treatment group (p = 0.76).
The median (ranges) sCD14 levels in NR patients were 1602.5 (1007.5–3310)
and 1547.5 (1082.5–3305) in R patients.During the IFN-based treatment, sCD14 levels slightly decreased without
statistically significant differences in both R and NR patients. The median
(ranges) sCD14 levels were the following in R: 1547.5 (1082.5–3305) at T0,
1335 (956–1677.5) at T1, 1132.5 (842.5–2085) at T2 (p>0.05). The median
(ranges) sCD14 levels in NR were the following: 1602.5 (1007.5–3310) at T0,
1214.25 (832.5–1852.5) at T1, 1246 (832.5–2612.5) at T2 (p>0.05). Also in
patients receiving IFN-free DAA regimens, there was no significant decrease
in sCD14 levels. The median (ranges) sCD14 levels were the following: 1778.9
(1092.5–2612.5) at T0, 1587.8 (552.5–2940) at T1, 1613.3 (1484.3–2347.5) at
T2 (p>0.05) (Fig
2).
Different impact of anti-HCV regimens on soluble biomarkers
In order to assess if IFN-based triple regimen or IFN-free DAA therapy exhibit a
different effect on CXCL10, sCD163 and sCD14 levels, we calculated for each
biomarker the reduction rate at T1 and T2 versus baseline (Fig 3A–3C). We observed a significant greater
reduction rate for CXCL10 during IFN-free therapies, in comparison to IFN-based
treatments. CXCL10 reduction rate at T1 and T2 was 60% vs 14% and 66% vs 31%,
respectively (Fig 3A); for
sCD163 it was 18% vs 11% and 16% vs 27%, respectively (Fig 3B); for sCD14 23% vs 7% and 1% vs 29%,
respectively (Fig 3C).
Fig 3
Different impact of anti-HCV regimens on soluble biomarkers.
Box plots represent the reduction rate of CXCL10 (panel A), sCD163 (panel
B) and sCD14 (panel C) at T1 and T2 versus T0. Box plots show
10th, 50th (median), 90th,
percentile and whiskers. Horizontal bars represent the median values.
Statistical differences were assessed between IFN-based versus IFN-free
treatment group at T1 and T2, by Mann-Whitney test. T0: baseline before
therapy; T1: after 4 weeks of therapy; T2: 12 weeks after the end of
treatment for R patient.
Different impact of anti-HCV regimens on soluble biomarkers.
Box plots represent the reduction rate of CXCL10 (panel A), sCD163 (panel
B) and sCD14 (panel C) at T1 and T2 versus T0. Box plots show
10th, 50th (median), 90th,
percentile and whiskers. Horizontal bars represent the median values.
Statistical differences were assessed between IFN-based versus IFN-free
treatment group at T1 and T2, by Mann-Whitney test. T0: baseline before
therapy; T1: after 4 weeks of therapy; T2: 12 weeks after the end of
treatment for R patient.
Biomarkers normalization in HCV-infected patients with sustained virological
response
To assess the degree of normalization of the three biomarkers at SVR12, we
compared the levels of CXCL10, sCD163 and sCD14 at T2 (SVR12) with controls HD.
Despite a decrease in concentrations, CXCL10 and sCD163 levels did not reach
normal values and remained significantly higher than in HD (p = 0.001 for CXCL10
and p<0.0001 for sCD163) (Fig 4A
and 4B). On the other hand, sCD14 levels at SVR12 were comparable
with those of HD (p>0.05) (Fig
4C).
Fig 4
CXCL10, sCD163 and sCD14 normalization in HCV-infected patients with
sustained virological response.
CXCL10 (panel A), sCD163 (panel B) and sCD14 (panel C) in HCV-infected
patients at T0, T2 and in HD. CXCL10 (panel D), sCD163 (panel E) and
sCD14 (panel F) in treated HCV-infected patients according to liver
fibrosis degree at T2 and in HD. Horizontal bars represent the median
values and horizontal dashed line in panel D, E and F indicates the
median values of HD. Kruskal-Wallis ANOVA with Dunn’s test were
performed to assess statistical differences between the different
groups. T0: baseline before therapy; T2: 12 weeks after the end of
treatment for R patient; F2-F0: mild to absence of fibrosis; F3: severe
fibrosis; F4: advanced fibrosis; HD: healthy donors; NS: not
significant.
CXCL10, sCD163 and sCD14 normalization in HCV-infected patients with
sustained virological response.
CXCL10 (panel A), sCD163 (panel B) and sCD14 (panel C) in HCV-infectedpatients at T0, T2 and in HD. CXCL10 (panel D), sCD163 (panel E) and
sCD14 (panel F) in treated HCV-infectedpatients according to liver
fibrosis degree at T2 and in HD. Horizontal bars represent the median
values and horizontal dashed line in panel D, E and F indicates the
median values of HD. Kruskal-Wallis ANOVA with Dunn’s test were
performed to assess statistical differences between the different
groups. T0: baseline before therapy; T2: 12 weeks after the end of
treatment for R patient; F2-F0: mild to absence of fibrosis; F3: severe
fibrosis; F4: advanced fibrosis; HD: healthy donors; NS: not
significant.Interestingly, after stratifying patients according to fibrosis stage, CXCL10
levels remained significantly higher, also in patients with low fibrosis,
compared to HD (Fig 4D).
Conversely, sCD163 levels decreased in patients with low fibrosis reaching
values comparable to those observed in HD (Fig 4E), even if the baseline sCD163 levels
were extremely higher than those measured in controls (1194, 790.6–2201.7 and
457.6, 279.2–810.5, p<0.0001). Regarding sCD14, plasma levels remained
significantly higher only in cirrhotic patients (Fig 4F), who showed elevated levels also at
baseline. Moreover, subjects with F0-F2 and F3 fibrosis, who showed only a
moderate increase of sCD14 at baseline, had normal levels in comparison with HD
at SVR12.
Discussion
In this study, we analyzed the temporal modifications of biomarkers of inflammation
and immune activation in a cohort of HCV-infectedpatients undergoing DAA therapies
using both IFN-based and IFN-free regimens. The circulating levels of CXCL10, sCD163
and sCD14 were assessed at baseline and during the course of treatment and results
were analyzed in terms of virological outcome and response to therapy. As previously
reported [19], we found
higher plasma concentrations of the three biomarkers at baseline in comparison to
controls suggesting systemic immune activation and inflammation, even in the low
fibrosis stages of HCV-related disease.CXCL10 is a member of the CXC subfamily of chemokines induced in monocytes,
fibroblasts, and endothelial cells by IFN-γ [4]. After binding to its receptor CXCR3, CXCL10
acts as a chemoattractant for CXCR3+ cells such T lymphocytes, monocytes and NK
cells. Intrahepatic and peripheral levels of CXCL10 are elevated in HCV-infectedpatients with high levels of liver inflammation and fibrosis [20]. The expression of CXCL10 mRNA by
hepatocytes correlates with serum CXCL10 levels [21] suggesting that CXCL10 may be a valid
surrogate marker of innate immune response activation and of ISG activation in the
liver [22]. During HCV
infection, T-helper 1 (Th1) lymphocytes secrete cytokines, such as IFN-γ and IL-2,
which activate monocytes and macrophages [23]. This immune reaction leads to liver tissue
damage and consequent progressive liver disease [24]. In the present study, CXCL10 levels
significantly decreased in patients reaching SVR12, suggesting that the decline of
this chemokine could be an indicator of disruption of the intrahepatic virus-host
interaction. Our study is in accordance with Romero A.I. et al. 2006, who showed a
reduction in responders after 6 weeks of IFN/RBV therapy [13]. Indeed, we found a decrease of CXCL10
levels in SVR patients after only one month of therapy, concurrently with HCV
suppression. On the other hand, the increased production of CXCL10 in NR patients
who stopped treatment for failure or adverse effects may be driven by the rebound of
viral replication.In a recent study, successful treatment with daclatasvir and asunaprevir decreased
serum levels of the ISG products CXCL10 and CXCL11, together with a decrease in
STAT1 expression and STAT1 phosphorylation in NK cells [25]. In our R patients, we also compared the
different impact of the IFN-based and IFN-free regimens on the degree of CXCL10
reduction. Although in R patients the two regimens were both effective in terms of
control of HCV replication, a significantly greater reduction of CXCL10 levels was
found in the IFN-free than in the IFN-based treatment group. Nevertheless, despite
the significant reduction in CXCL10 during treatment, the levels did not reach
normal values, suggesting the persistence of residual inflammation. In our study
population, we could not observe a significant difference in CXCL10 levels between
patients with advanced fibrosis and in those with mild fibrosis, as
reported by Diago M. et al. [26], thus underlying the presence of immune activation even in the low
fibrosis stages. However, in accordance with Romero A.I. et al [13], we found a positive
correlation between CXCL10 levels and fibrosis index (FIB-4), suggesting a possible
role of CXCL10 as a non-invasive marker of liver fibrosis.Furthermore, we studied two markers of monocyte activation, sCD163 and sCD14, which
contribute to hepatic inflammation and fibrosis. sCD163 is an important marker of
macrophage activation, involved in the activation of hepatic stellate cells during
the progression of chronic liver disease [27,28]. In our study, we found higher levels of
sCD163 in HCV-infectedpatients compared to HD, confirming that sCD163 plays an
important role in the pathogenesis of HCV infection [29]. In accordance with Kazankow K. et al.
[16], we found higher
levels of sCD163 in patients with advanced fibrosis (F4) compared to patients with
mild fibrosis (F0-F2), showing that sCD163 could play a role in determining the
severity of hepatitis. Furthermore, in our study sCD163 levels correlated with
fibrosis assessments (FIB-4 and stiffness), suggesting that sCD163 is a promising
fibrosis marker during HCV infection [30]. Moreover, the increase in monocyte
activation markers has been associated with several cardiovascular and
neurodegenerative diseases, such as subclinical carotid artery disease in general
population and in HCV or HIV infected subjects [31-34], suggesting a possible extra-hepatic effect
of HCV, mediated by monocyte activation.During anti-HCV therapies, sCD163 levels significantly decreased in R patients
undergoing both IFN-based or IFN-free regimens, while they remained high in NR
patients who stopped IFN-based treatments for failure or adverse effects. Comparing
the two treatment strategies, even if the rate of reduction was lower at T1 in the
IFN-based treatment group, no statistically significant differences were observed
between the two regimens. Furthermore, in patients who were treated with IFN-based
therapy, significantly higher baseline sCD163 levels were observed in NR patients
compared to R patients, while in the IFN-free group all patients obtained SVR,
including those with higher levels of sCD163 at baseline. Although all patients were
treated with DAAs, the response to first generation DAA protease inhibitors was
still dependent on the immunomodulating effect of INFα. Comparing inflammation
markers reduction rate, we observed a less marked decrease in sCD163 than in CXCL10
plasma levels. Moreover, patients obtaining SVR12 still showed higher levels of
sCD163 when compared to healthy subjects. In a recent study sCD163 and Mac2BP levels
didn’t normalize within 6 months from the start of interferon-free therapy [35]. Interestingly, in our
study, only patients with low fibrosis showed a normalization of sCD163 plasma
levels. These findings underline the need to start therapy for HCV infection in an
early stage of fibrosis in order to block HCV-induced inflammation. In fact, the
persistence of a systemic inflammatory response could be responsible of an increased
risk of inflammatory-related organ diseases in HCV infected subjects.Finally, systemic inflammation associated with gut derived microbial products has
been described during HCV infection [17]. sCD14 is a glycosyl phosphatidyl inositol that is expressed on
monocytes, macrophages, polymorphonuclear leukocytes and dendritic cells [36]. CD14 is a marker of
microbial translocation and is secreted in a soluble form upon monocyte activation.
It was demonstrated that sCD14 could be detected at high levels in patients with HCV
infections [36] and recent
studies suggested a relationship between microbial translocation and progression of
liver disease during HCV infection [17]. However, elevated levels of sCD14 have also been observed in
patients with non-alcoholic steatohepatitis [37], and common variable immunodeficiency
[38], suggesting other
possible origins of sCD14 in plasma, one of which may be the liver.In our study population, we found a moderate increase in sCD14 with a significant
increase only in patients with F4 liver fibrosis stage, as described previously
[17]. In prior work
[39] no significant
changes were found in sCD14 levels in patients with HCV infection during
PEG-IFNα/RBV therapy, whereas a recent study showed an increase in innate immune
activation, measured as sCD14 and IL-18 elevation [40]. In this study the authors showed that a
group of patients with low baseline levels of these factors, had a very high SVR
rate during the course of dual therapy.We found that sCD14 levels remained apparently unchanged both in NR and R patients at
the end of therapy, despite the control of HCV viremia with no differences between
the two therapies. Indeed, when comparing patients who obtained SVR12 with HD, sCD14
levels were similar, but after stratifying patients according to fibrosis stage,
cirrhotic subjects showed persistently higher levels of sCD14, indicating a lack of
normalization after HCV eradication.The main limitations of our research are the different clinical characteristics of
the two therapeutic groups in terms of liver fibrosis and HCV genotypes due to the
distinct indications in the national guidelines at the time of treatment. In fact,
current Italian guidelines for HCV treatment allow the use of new DAAs only in
patients with advanced fibrosis (F3-F4). Moreover, the number of subjects with low
liver fibrosis was limited, together with a short post-treatment follow-up
interval.In conclusion, our results suggest that the systemic levels of inflammatory
biomarkers in HCV-infected subjects were globally reduced by anti-HCV therapy, with
a more pronounced effect produced by IFN-free regimens on CXCL10 levels. A possible
explanation is that CXCL10 declines simoultaneously with the level of viremia and
the new and potent therapies with DAAs induce a rapid decrease in HCV replication
with a subsequent sudden reduction of CXCL10 plasma levels. This chemokine is
induced after RIG-I and TLR-3 activation by HCV RNA, which acts as a PAMP.
Furthermore CXCL10 belongs also to the IFN-stimulated gene family [41]. Thus, CXCL10 levels are
directly related to viral load, while the other inflammatory markers studied in our
work are secondary immune events. In fact, CXCL10 should be considered more linked
to viral induced inflammation, while sCD163 and sCD14 more related to secondary
immune events that lead to hepatic fibrosis. Moreover IFN-based therapy could induce
an immune activation due to the direct effect of IFN that counteracts the
anti-inflammatory role linked to HCV suppression.At the end of therapy, CXCL10 and sCD163 plasma levels remain significantly higher
than in HD. Probably the chronic exposure to viral antigens may cause exhaustion of
the immune system, resulting in a slow immunological recovery. The low degree of
immune recovery could also have important clinical implications that prompt a close
monitoring of immune-mediated organ disease associated to with HCV.
A recent study suggests that after SVR, T-cell function remains abnormal and
hyporesponsive in most cases probably because of cell exhaustion [42]. Only in patients with low
fibrosis a normalization of sCD163 was achieved, suggesting the need for an early
therapeutic approach during HCV infection. A longer follow-up could clarify if
inflammation will resolve over time or persist after HCV eradication.
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