Download Elevated HMGB1-related interleukin

Int J Clin Exp Pathol 2015;8(2):1341-1353
www.ijcep.com /ISSN:1936-2625/IJCEP0004798
Original Article
Elevated HMGB1-related interleukin-6 is associated with
dynamic responses of monocytes in patients with active
pulmonary tuberculosis
Jin-Cheng Zeng1,3*, Wen-Yu Xiang3*, Dong-Zi Lin2, Jun-Ai Zhang1, Gan-Bin Liu2, Bin Kong3, Yu-Chi Gao3,
Yuan-Bin Lu3, Xian-Jing Wu4, Lai-Long Yi2, Ji-Xin Zhong5, Jun-Fa Xu1,3
Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, 1 Xincheng Road, Dongguan
523808, People’s Republic of China; 2Dongguan 6th People’s Hospital, Dongguan 523008, People’s Republic
of China; 3Department of Clinical Immunology, Institute of Laboratory Medicine, Guangdong Medical College,
No. 1 Xincheng Road, Dongguan 523808, People’s Republic of China; 4Department of Clinical Laboratory,
Affiliated Hospital of Guangdong Medical College, Zhanjiang 524001, People’s Republic of China; 5Division of
Cardiovascular Medicine, University of Maryland School of Medicine, Maryland 21201, USA. *Equal contributors.
1
Received December 13, 2014; Accepted February 5, 2015; Epub February 1, 2015; Published February 15, 2015
Abstract: There were limited studies assessing the role of HMGB1 in TB infection. In this prospective study, we aimed
to assess the levels of HMGB1 in plasma or sputum from active pulmonary tuberculosis (APTB) patients positive
for Mtb culture test, and to evaluate its relationship with inflammatory cytokines and innate immune cells. A total of
36 sputum Mtb culture positive APTB patients and 32 healthy volunteers (HV) were included. Differentiated THP-1
cells were treated for 6, 12 and 24 hrs with BCG at a multiplicity of infection of 10. The absolute values and percentages of white blood cells (WBC), neutrophils, lymphocytes, and monocytes were detected by an automatic blood
analyzer. Levels of HMGB1, IL-6, IL-10 and TNF-α in plasma, sputum, or cell culture supernatant were measured by
ELISA. The blood levels of HMGB1, IL-6, IL-10 and TNF-α, the absolute values of WBC, monocytes and neutrophils,
and the percentage of monocytes were significant higher in APTB patients than those in HV groups (P<0.05). The
sputum levels of HMGB1, IL-10, and TNF-α were also significantly higher in APTB patients than those in HV groups
(P<0.05). Meanwhile, plasma level of HMGB1, IL-6, and IL-10 in APTB patients were positively correlated with those
in sputum (P<0.05), respectively. IL-6 was positively correlated with HMGB1 both in plasma and sputum of APTB
patients (P<0.05). HMGB1 and IL-6 is positively correlated with the absolute number of monocytes in APTB patients
(P<0.05). BCG induced HMGB1, IL-6, IL-10 and TNF-α production effectively in PMA-treated THP-1 cells. HMGB1
may be used as an attractive biomarker for APTB diagnosis and prognosis and may reflect the inflammatory status
of monocytes in patients with APTB.
Keywords: Active pulmonary tuberculosis, infection, cytokines, HMGB1, immunity
Introduction
Tuberculosis (TB) remains a global health problem. It has been estimated that 8.6 million individuals worldwide developed TB and more than
1.3 million died from TB-related disease, in
2012 [1, 2]. In China, TB infection incidence
has been estimated as 50~124 cases per
100,000 inhabitants per year [1]. The rapid and
accurate detection of TB still relies on sputum
smear microscopy and culture in a well-managed and equipped laboratory network,
although it takes weeks to the result. The prognosis of the TB disease depends on the ability
of the host to eliminate the mycobacterium
tuberculosis (MTB). In the removal process,
innate immune cells, adaptive immune cells
and inflammatory cytokines, etc. play important
roles.
HMGB1 (previously HMG1; HMG-1; HMG 1;
amphoterin; p30), an inflammatory cytokines,
functions in nucleus (e.g., nucleosome stability,
genome chromatinization, nuclear catastrophe,
DNA binding, V (D) J recombination, DNA replication, telomere stability and gene transcription), cytoplasm (e.g., autophagy, unconventional secretory pathway), membranes (e.g., neurite
outgrowth, platelet activation, cell differentiation, erythroid maturation, adhesion and innate
HMGB1 handling IL-6 and tuberculosis
immunity) and extracellular environment (e.g.,
inflammation, immunity, migration, invasion,
proliferation, differentiation, antimicrobial defense and tissue regeneration) [3-7]. Especially,
extracellular HMGB1, released by immune cells
(e.g., macrophages, monocytes, neutrophils,
DCs, NKs), fibroblasts, or epithelial cells, functions as a DAMP to alert the innate immune system by recruiting inflammatory cells in lung disease (e.g., asthma, COPD, acute respiratory
distress syndrome, cystic fibrosis, pneumonia,
acute lung injury and lung cancer) [3, 8-13].
Notably, extracellular HMGB1 also functions as
an immune adjuvant to trigger a robust
response of T cells, dendritic cells, and endothelial cells [3, 14-18]. It is well established
that HMGB1 as a pro-inflammatory cytokine in
many inflammatory diseases [3]. Extracellular
or blood HMGB1 levels were found increased in
patients with juvenile idiopathic arthritis, atrial
fibrillation, gastric cancer, and septic shock,
and closely associated with the clinical and
pathologic features [19-22]. In addition, suppression of HMGB1 release and/or activity significantly decreased the inflammatory response, tissue injury, and death in animals [3,
23-25]. These properties of HMGB1 make it an
attractive biomarker and therapeutic target.
Therefore, there are many translational studies
ongoing to assess the feasibility of HMGB1 suppression as therapeutic strategies for inflammatory diseases [3, 13, 26-28].
Recently, Grover et al. reported that HMGB1,
independent of its interaction with receptor for
advanced glycation end products (RAGE),
enhanced the protective efficacy and cellular
immune response of TB subunit vaccines as an
adjuvant [29]. Currently, only limited information is available about the role of HMGB1 in TB
infection. Hence, in this prospective study, we
aimed to assess whether HMGB1 is detectable
or mutative both in plasma and sputum of
active pulmonary tuberculosis (APTB) patients
positive for Mtb culture test, and to evaluate its
relationship with inflammatory cytokines (e.g.,
IL-6, IL-10 and TNF-α) and innate immune cells
(e.g., monocytes and neutrophils).
Subjects and methods
Subjects
A total of 36 sputum Mtb culture positive APTB
patients and 32 healthy volunteers (HV) were
recruited from April 2013 to April 2014 in
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Dongguan 6th People’s Hospital (Dongguan,
China) with the following inclusion/exclusion
criteria [30]. All subjects were MTB positive in
sputum culture test. Age, gender, tuberculin
skin test, fever and smoking status were
obtained at the screening evaluation. Subjects
with HIV infection, diabetes, cancer, autoimmune diseases, immunosuppressive treatment, or pulmonary tuberculosis history were
excluded from the study. The study was
approved by the Internal Review and the Ethics
Boards of Guangdong Medical College and
Dongguang 6th People’s Hospital, and informed
consent was obtained from all study subjects.
Plasma and sputum preparation
After registering eligible, patients were asked to
rinse their mouth with water and bloods and
sputum concomitantly samples were collected.
6 mL blood was drawn into anticoagulated
tubes (Improve medical, Guangzhou, china).
One third of the blood samples were used to
analyze counts and percentages of WBC, neutrophils, lymphocytes, and monocytes by an
automatic blood analyzer (UniCel DxC 800
Synchron, Beckman coulter, California, USA).
Remaining blood samples were centrifuged at
1500 rpm for 15 min, and plasma samples
were collected and stored at -80°C until analysis. Induced sputum collection was done 2 hrs
later. After brushing teeth and rinsing mouth,
patients were instructed to inhale sterile 3%
saline for up to 30 min via a disposable sputum
suction device (Hangzhou Medtec Medical
Devices, Hangzhou, China) completed by nurses. Sputum samples were collected directly
into 50 ml sterile disposable polypropylene
centrifuge tubes (Corning incorporated, NY,
USA) and processed within 2 hrs of collection
as described previously [31] with minor modifications. Briefly, sputum was diluted with an
equal amount of Hank’s solution containing
0.1% dithiothreitol and incubated on a rocking
platform for 15 min at room temperature to
digest mucus. Following digestion, half of the
sputum digests were removed for AFB smear
and quantitative culture. Remaining sputum
digests were centrifuged at 1500 rpm and filtered with 0.22 μm disposable filters (Millipore,
MA, USA) to sediment cellular or bacterial constituents and stored at -80°C until analysis.
Sputum specimens from healthy control subjects (induced sputum only) were collected and
processed following the same procedure as
that of APTB patients.
Int J Clin Exp Pathol 2015;8(2):1341-1353
HMGB1 handling IL-6 and tuberculosis
Table 1. Demographics of subjects included in the study
Groups
APTB
HV
t/x
P
n
36
32
Age, years
38.550±1.610 37.210±1.520 0.6010 >0.05
Male/Female
20/16
17/15
0.040 >0.05
Smoking (ever), n (%)
5 (13.9)
3 (9.4)
New/Relapsed
28/8
TST, ≥10 mm, n (%)
36 (100)
0 (0)
AFB Smear-positive, n (%)
36 (100)
0 (0)
Fever, n (%)
0 (0)
0 (0)
2
TST = Tuberculin skin test; AFB = Acid-fast bacilli.
Infection of THP-1 cell line with BCG
M. bovis BCG was obtained from Zheng W.
Chen laboratories, University of Illinois College
of Medicine, Chicago, Illinois, USA. Mycobacterial strains were grown to mid-log phase
in MB7H9 medium supplemented with albumin-dextrose-catalase (1×), glycerol (0.5%) and
Tween 80 (0.05%) according to the method
described by Huang et al [32]. PBS stocks were
prepared and stored at -80°C until further use.
The CFU of stocks was enumerated by plating
appropriate dilutions in duplicates on MB7H11
agar supplemented with oleic acid-albumindextrose-catalase (1×) and glycerol (0.5%) [32].
THP-1 cells were obtained from cell bank of
Chinese Academy of Sciences, Shanghai,
China, and were maintained in RPMI 1640
medium supplemented with 10% fetal bovine
serum, 100 units/ml penicillin, and 100 μg/ml
streptomycin at 37°C in a humidified incubator
with 5% CO2. The cells were seeded in 6-well
microplates at a concentration of 1×106 cells/
well followed by adding 100 nM PMA for 24 hrs
to induce the cells differentiation into macrophages. Differentiated THP-1 cells were treated
for 6, 12 and 24 hrs with heat-inactivated BCG
at a multiplicity of infection of 10. 1 μg/ml lipopolysaccharide (LPS) was used as positive control and PBS was used as negative control.
After culture for an indicated period, cell culture
supernatants were collected and filtered
through a 0.22 μm filter, and stored at -80°C
until analysis.
ELISA detection
Cytokines were measured in the plasma, sputum and cell culture supernatant samples using
human HMGB1 ELISA Kit (Bluegene Biotech,
Shanghai, China) and human IL-6, IL-10 and
TNF-α ELISA Kit (RayBiotech, Atlanta, USA)
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according to the manufacturer’s instructions. Cell culture supernatant was diluted 1:10. Plasma and processed sputum were used
as an undiluted specimen.
Statistical analysis
Normality test was first performed to decide whether
the data were in normal distribution. Data were shown
in Mean ± SEM (standard
error of the mean, SEM). Student’s t-test or
ANOVA (analysis of variance, ANOVA) were
employed to compare the differences of measured data, and Pearson correlation was used
to measure the degree of dependency between
variables by the GraphPad Prism version 5.0
software (GraphPad Software Inc., San Diego,
CA, USA). A P value of less than 0.05 was considered as statistical significant.
Results
Characteristics of the subjects included in the
study
We prospectively enrolled 68 subjects. Among
them, 36 cases were active pulmonary tuberculosis (APTB) and 32 cases were healthy volunteers (HV). The demographic and clinical characteristics for all study subjects are shown in
Table 1. No significant difference in terms of
age and gender was noted between APTB and
HV groups. Among APTB patients, 100%
(36/36) of them were MTB positive by sputum
smear analysis, 77.8% (28/36) of them were
newly APTB patients, and 100% (36/36) of
them were positive for tuberculin skin test (TST)
(induration diameter ≥10 mm).
HMGB1, IL-6, IL-10 and TNF-α are increased
both in plasma and sputum of APTB patients
It has been reported that during lung disease
like asthma, COPD, acute respiratory distress
syndrome, cystic fibrosis, pneumonia and lung
cancer, blood HMGB1 levels are increased and
correlates with disease severity [3, 8-13].
Additionally, HMGB1 production is increased in
sputum and contributes to inflammatory
response and lung matrix degradation in cystic
fibrosis airway disease [11, 33]. Therefore, we
evaluated the level of HMGB1 in both plasma
Int J Clin Exp Pathol 2015;8(2):1341-1353
HMGB1 handling IL-6 and tuberculosis
Figure 1. Abundance of HMGB1, IL-6, IL-10 and TNF-α both in plasma and sputum of APTB patients. ELISA was
performed in concomitantly collected plasma and sputum samples. Plasma and sputum HMGB1 (A), IL-6 (B), IL-10
(C) and TNF-α (D) were significantly increased in APTB patients (n=36) compared to HV (n=32). Data were shown as
Mean ± SEM. *, P<0.05; **, P<0.01; ***, P<0.0001.
and sputum, in 36 APTB patients and 32 HV
subjects. At the same time, levels of IL-6, IL-10
and TNF-α in plasma and sputum were also
analyzed to evaluate inflammatory response in
blood and airways or lung parenchyma.
patients. However, sputum level of IL-16 (-1.58fold) was significantly lower than that in plasma
in APTB patients. No significant differences in
these cytokines were found between plasma
and sputum HV groups (Figure 1A-D).
Results showed that plasma level of HMGB1
(1.68-fold) of APTB patients were significantly
higher than those in HV groups (Figure 1A). We
also found increased plasma levels of IL-6
(1.86-fold), IL-10 (1.44 folds) and TNF-α (1.63fold) in APTB patients (Figure 1B-D), which was
consistent with previously published studies on
TB patients [34, 35]. Notably, sputum levels of
HMGB1 (1.82-fold), IL-10 (1.59-fold), and TNF-α
(2.18-fold) were significantly higher than those
in HV groups (Figure 1A-D). Interestingly, levels
of HMGB1 (1.27-fold), IL-10 (1.30-fold), and
TNF-α (1.50-fold) were also significantly higher
in sputum than those in plasma in APTB
These results suggested that HMGB1, IL-10,
and TNF-α may be involved in a dynamic inflammatory response to TB infection in blood and
airways (or lung parenchyma). Especially in airways, released HMGB1, IL-10, and TNF-α may
be a useful marker for assessing the airways or
lung parenchyma inflammatory response to TB
infection.
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Plasma level of HMGB1, IL-6 and IL-10 in APTB
patients are positive correlate to themselves in
sputum, respectively
We further investigated correlations of plasma
levels of HMGB1, IL-6, IL-10, and TNF-α with
Int J Clin Exp Pathol 2015;8(2):1341-1353
HMGB1 handling IL-6 and tuberculosis
These results suggested that elevated level of HMGB1, IL-6, and
IL-10 in plasma may be closely
related to the activity of inflammatory response caused by TB infection. They also suggested HMGB1,
IL-6, and IL-10 may share some
similar response pathway to TB
infection, although the abundance
of IL-6 levels between plasma and
sputum was inconsistent with the
abundance of HMGB1 and IL-10
(Figure 1).
IL-6 positively correlates with
HMGB1 in both plasma and sputum of APTB patients
Figure 2. Relationship between the plasma and sputum levels of
HMGB1, IL-6, IL-10 and TNF-α in APTB patients and HV subjects. Correlation of plasma and sputum level of HMGB1, IL-6, IL-10 and TNF-α
in APTB patients (A) and HV subjects (B) were analysised by Pearson
correlation using GraphPad Prism Version 5.0. Plasma levels of HMGB1
(A-a), IL-6 (A-b) and IL-10 (A-c), but not TNF-α (A-d) in APTB patients were
positively correlated with those in sputum, respectively. However, plasma levels of HMGB1 (B-a), IL-6 (B-b), IL-10 (B-c) and TNF-α (B-d) in HV
subjects did not correlate with those in sputum.
those in sputum, in either APTB patients or HV
groups (Figure 2A and 2B). Interestingly, plasma levels of HMGB1, IL-6, and IL-10, but not
TNF-α, were positively correlated with those in
sputum after TB infection (Figure 2A and 2B).
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In order to explore the relationship
among the inflammatory cytokines, correlations of HMGB1, IL-6,
IL-10, and TNF-α levels with each
other were analyzed in either plasma or sputum (Figure 3A and 3B).
Consistent with previously published studies in disease of severe community-acquired infections
and bacteraemia [36], plasma
level of IL-6 showed a positive correlation with HMGB1 in this study
(Figure 3A-a). Furthermore, in line
with the findings by Nemeth, et al.
[37], there was a positive correlation between plasma level of IL-6
and plasma level of TNF-α in APTB
patients (Figure 3A-b). Interestingly, similar to the findings in plasma, sputum level of IL-6 displayed
a positive correlation with sputum
level of HMGB1 and TNF-α (Figure
3B-a, 3B-b) respectively. While in
plasma or sputum of APTB
patients, level of HMGB1 had no
correlation with level of IL-10
(Figure 3A-c, 3B-c) or level of
TNF-α (Figure 3A-d, 3B-d), and
level of IL-10 also had no correlation with level of TNF-α (Figure
3A-e, 3B-e) or level of IL-6 (Figure
3A-f, 3B-f).
These results further indicated that HMGB1
and IL-6 may share some similar or the same
response pathway to TB infection. The above
results also indicated the levels of different
cytokines are fine-tuned and the delicate balInt J Clin Exp Pathol 2015;8(2):1341-1353
HMGB1 handling IL-6 and tuberculosis
Figure 3. Relationship among the levels
of HMGB1, IL-6, IL-10 and TNF-α in plasma and sputum of APTB patients. Correlations among the levels of HMGB1,
IL-6, IL-10 and TNF-α in plasma (A)
and sputum (B) in APTB patients were
analyzed by Pearson correlation using
GraphPad Prism Version 5.0. Plasma
level of HMGB1 was positively correlated with plasma levels of IL-6 (A-a),
and plasma level of TNF-α was positively correlated with plasma level of
IL-6 (A-b). There were no significant correlations between plasma HMGB1 level
and plasma IL-10 (A-c), between plasma
HMGB1 level and plasma TNF-α (A-d),
between plasma TNF-α and plasma IL10 (A-e), and between plasma IL-6 and
plasma IL-10 (A-f). Similarly, sputum level of HMGB1 was positively correlated
with sputum level of IL-6 (B-a), and sputum level of TNF-α was positively correlated with sputum level of IL-6 (B-b).
There were no signification correlations
between sputum HMGB1 and sputum
IL-10 (B-c), between sputum HMGB1
and sputum TNF-α (B-d), between sputum TNF-α and sputum IL-10 (B-e), and
between sputum IL-6 and sputum IL-10
(B-f).
ance between the pro-inflammatory
and anti-inflammatory cytokine signaling is well established at APTB
progression.
HMGB1 handling IL-6 may be associated with monocytes dynamic
responses in APTB patients
It is clear that HMGB1 plays a critical role in the regulation of innate
and adaptive immune responses
[3, 37]. Both innate and adaptive
immunity play important roles in
the host defense against MTB
infection [38-43]. In accordance
with the findings by Veenstra, et al.,
we found the absolute WBC, monocytes, and neutrophils counts were
significantly higher, but lymphocytes were significantly lower in
APTB patients compared to those
in HV groups (Table 2). Meanwhile,
the percentage of monocytes was
significantly higher, but lymphocytes were significantly lower in
APTB patients (Table 2). It’s worth
noting that the absolute value of
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Int J Clin Exp Pathol 2015;8(2):1341-1353
HMGB1 handling IL-6 and tuberculosis
Table 2. Blood cell differential of subjects (Mean ± SEM)
Groups
APTB (n=36)
HV (n=32)
t
P
9
WBC, ×10 /L
7.531±0.313
6.014±0.310
3.430 <0.01
Mono, ×109/L 0.4561±0.0282 0.3419±0.02611 2.949 <0.01
Lymp, ×109/L
1.337±0.221
2.031±0.209
2.266 <0.05
9
Neut, ×10 /L
5.360±0.330
4.094±0.368
2.569 <0.05
Mono, %
7.533±0.683
5.259±0.541
2.525 <0.05
Lymp, %
20.216±2.133
30.899±2.744 3.109 <0.01
Neut, %
60.310±3.852
51.630±3.728 1.610 >0.05
ing 12~24 hrs after BCG (1.7-fold)
or LPS (4.9-fold) stimulation (Figure 5C). In addition, the baseline
TNF-α production in PMA-treated
THP-1 cells was at a high level, and
both BCG and LPS stimulation
showed a near 2-fold increase at
24 hrs (Figure 5D).
These results suggested that monocytes like macrophage generatWBC = white blood cells; Mono = monocytes; Lymp = lymphocytes; Neut =
ed HMGB1, IL-6, IL-10, and TNF-α
neutrophils. Groups were compared by t test with GraphPad Prism Version
after TB infection, but who was the
5.0.
instigator is unknown. These results also further supported HMGblood monocytes was positively correlated to
B1 handling IL-6 may be associated with monoplasma levels of HMGB1 (Figure 4A) and IL-6
cytes dynamic responses in APTB patients. A
(Figure 4B), but not IL-10 (Figure 4C) or TNF-α
recent report showing anti-IL-6 protein inhibits
(Figure 4D).
HMGB1 by macrophage also supported our
hypothesis [44].
These results suggested that HMGB1 handling
IL-6 may be associated with monocytes dynamDiscussion
ic responses in APTB patients and also indicatThe innate cells including macrophages, monoed that the elevation of plasma level of HMGB1
cytes, dendritic cells (DCs), mast cells, neutroand IL-6 may be directly related to the elevation
phils, eosinophils and natural killer cells were
of blood monocytes.
the majority producers of HMGB1, IL-10, TNF-α
BCG induced HMGB1, IL-6, IL-10, and TNF-α
and IL-6 in pulmonary infection [3, 45-47]. In
production in PMA-treated THP-1 cells
this study, for the first time to our knowledge,
we found that HMGB1 is detectable both in
To better address the relationship of HMGB1,
plasma and sputum of patients positive for
IL-6, IL-10 and TNF-α production with monosputum Mtb culture test and it was increased in
cytes dynamic responses after TB infection,
comparison with healthy volunteers. Moreover,
THP-1 cells that differentiated into a macroelevated levels of IL-6, IL-10 and TNF-α were
phage-like phenotype following the treatment
also detected on the corresponding subjects in
with 100 nM PMA for 24 hrs were used to incuplasma and sputum. The blood levels of IL-6,
bate with BCG at a multiplicity of infection of 10
IL-10 and TNF-α had been shown to increase in
for 6, 12 and 24 hrs. HMGB1, IL-6, IL-10 and
patients with TB infection [34-37]. Levels of
TNF-α were detected in the cell culture superTNF-α and IL-10 were elevated in sputum from
natant. LPS was used as a positive control and
TB patients, indicating that their production
PBS was served as a negative control. As shown
may be a dynamic response associated with
in Figure 5, BCG in accordance with LPS
pulmonary infection [48, 49]. In contrast to the
induced HMGB1, IL-6 and TNF-α, but not IL-10
findings of TNF-α and IL-10 described above,
production from PMA-treated THP-1 cells in a
level of IL-6 in sputum did not follow the same
time-dependent manner. HMGB1 production,
pattern as IFN-a, although TNF-α and IL-6 may
at 24 hrs, showed 4-fold increased after BCG
be produced by macrophages and neutrophils,
stimulated, and near 8-fold increased after LPS
both of which represented the majority of cells
stimulation in comparison with un-stimulated
found in sputum from TB patients and healthy
group (Figure 5A). In accordance with HMGB1,
volunteers [50].
IL-6 production at 24 hrs showed more than
2-fold increase after BCG stimulation and more
Interestingly, level of HMGB1, IL-10 and TNF-α,
than 9-fold increase after LPS stimulation
but not IL-6, were significantly higher in sputum
(Figure 5B). However, IL-10 reached the highest
than those in plasma in APTB patients, suglevel at 12 hrs after BCG (2.4-fold) or LPS (7.5gesting HMGB1, IL-10, TNF-α but not IL-6 may
fold) stimulation and gradually decreased durdisplay a more dynamic inflammatory response
1347
Int J Clin Exp Pathol 2015;8(2):1341-1353
HMGB1 handling IL-6 and tuberculosis
Figure 4. Relationship between the plasma level of HMGB1, IL-6, IL-10 and TNF-α to the absolute values of blood
monocytes (Mono) in APTB patients. Correlations of the absolute value of blood monocytes (Mono) in APTB patients
with plasma levels of HMGB1 (A), IL-6 (B), IL-10 (C) and TNF-α (D) were analyzed by Pearson correlation using GraphPad Prism Version 5.0. The absolute value of blood monocytes were positively correlated to plasma levels of HMGB1
(A) and IL-6 (B), but not IL-10 (C) and TNF-α (D).
to TB infection in airways or lung parenchyma
than blood. To better address the role of
HMGB1 in the pathogenesis of TB infection, the
correction of HMGB1 level between plasma
and sputum, and correlations of HMGB1 with
other inflammatory cytokines or blood innate
immune cells were further evaluated. We
showed that HMGB1 level was positively correlated with IL-6 level both in plasma and sputum
of APTB patients, at the same time, both
HMGB1 and IL-6 were associated with mono-
1348
cytes, but not with neutrophils or lymphocytes
(Dates not shown) dynamic responses in APTB
patients. These results suggested that HMGB1
may handle IL-6 to involve in a monocytes
dynamic inflammatory response to TB infection.
Previous studies have demonstrated HMGB1
mediated the inflammatory response to initiate
by TLRs (TLR2, TLR4 and TLR9) in neutrophils,
monocytes, and macrophages. And IL-6 de-
Int J Clin Exp Pathol 2015;8(2):1341-1353
HMGB1 handling IL-6 and tuberculosis
Figure 5. Abundance of HMGB1, IL-6, IL-10 and TNF-α in supernatant of THP-1 cells after BCG infection. PMA differentiated THP-1 cells were infected with BCG. Infection was continued for 6, 12 and 24 hrs and supernatant was
collected for the detection of HMGB1, IL-6, IL-10 and TNF-α. LPS was used as a positive control and PBS was used
as a negative control. The values and error bars represent average and standard error of the mean (SEM) of three
independent set of experiments. *, P<0.05; **, P<0.01; ***, P<0.0001.
pends on a TLR4/MyD88-dependent pathway
involving p38 MAPK and NF-κB [46, 51, 52].
Therefore, HMGB1 handling IL-6 may be associated with monocytes dynamic responses after
TB infection.
To better address that HMGB1 and IL-6 could
co-produce by monocytes, a PMA-treated
THP-1 cells displaying a macrophage-like phenotype were used. Results showed that HMGB1
and IL-6, unlike IL-10 and TNF-α, had a similar
generation process in PMA-treated THP-1 cells
after BCG infection. The study of the interaction
1349
between HMGB1 and IL-6 has been reported in
other disease and models. Plasma level of
HMGB1 positively correlated with plasma level
of IL-6 in patients with suspected communityacquired infections with sepsis, severe community-acquired infections with bacteraemia, neonatal sepsis model, but not in patients with
septic shock, patients with severe sepsis and
septic shock, patients with bacteraemia only,
burned patients, children with febrile seizures,
and mouse model of polymicrobial sepsis [36,
53-59]. In addition, anti-IL-6R antibody prevented the early loss of transplanted mouse islets
Int J Clin Exp Pathol 2015;8(2):1341-1353
HMGB1 handling IL-6 and tuberculosis
in parallel with reduced HMGB1 production by
hepatic mononuclear cells [44]. In addition, IL-6
and HMGB1 may form a positive feedback loop
to amplify the downstream signals. The concentration of HMGB1 increased as early as the
elevation of IL-6 in reducing renal ischemiareperfusion injury (IRI) [60]. HMGB1 neutralization enhanced hepatic TNF-α, IL-6 mRNA
expression during acetaminophen hepatotoxicity [61]. And the L. sanguineus (Ls) IL-6 transcription was significantly induced by recombinant LsHMGB1 in HK leukocytes [62]. These
results indicated that HMGB1 may play an
important role in immune response by positive
feedback to IL-6.
In conclusion, for the first time to our knowledge we showed that the plasma and sputum
levels of HMGB1 were significantly increased in
MTB infected patients in comparison with
healthy donors and significantly correlated with
IL-6 level, a pro-inflammatory cytokines,
involved in a monocytes dynamic inflammatory
response to against TB infection, although the
correlation is low.
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
Acknowledgements
This work was supported by the National Natural Science Foundation of China (30971779, 81101553, 81273237), the Key Project
of Science and Technology Innovation of Education Department of Guangdong Province
(2012KJCX0059), the Science and Technology
Project of Dongguan (201450715200503), the
Science and Technology Project of Zhanjiang
(2013C03012) and the Science and Technology
Innovation Fund of GDMC (STIF201110, B2012078, M2013046).
[10]
[11]
Disclosure of conflict of interest
None.
Address correspondence to: Dr. Jun-Fa Xu, Guangdong Provincial Key Laboratory of Medical Molecular
Diagnostics, No. 1 Xincheng Road, Dongguan
523808, People’s Republic of China. E-mail: [email protected]; [email protected]
References
[1]
Zumla A, George A, Sharma V, Herbert N, Baroness MO. WHO’s 2013 global report on tuberculosis: successes, threats, and opportunities.
Lancet 2013; 382: 1765-1767.
1350
[12]
[13]
WHO. Global tuberculosis report 2013. Geneva: World Health Organization; 2013.
Kang R, Chen R, Zhang Q, Hou W, Wu S, Cao L,
Huang J, Yu Y, Fan XG, Yan Z, Sun X, Wang H,
Wang Q, Tsung A, Billiar TR, Zeh HJ 3rd, Lotze
MT, Tang D. HMGB1 in health and disease. Mol
Aspects Med 2014; 40C: 1-116.
Jessop F, Holian A. Extracellular HMGB1 regulates multi-walled carbon nanotube-induced
inflammation in vivo. Nanotoxicology 2014;
[Epub ahead of print].
Wang HL, Peng LP, Chen WJ, Tang SH, Sun BZ,
Wang CL, Huang R, Xu ZJ, Lei WF. HMGB1 enhances smooth muscle cell proliferation and
migration in pulmonary artery remodeling. Int J
Clin Exp Pathol 2014; 7: 3836-3844.
Yanai H, Taniguchi T. Nucleic acid sensing and
beyond: virtues and vices of HMGB1. J Intern
Med 2014; 276: 444-453.
Magna M, Pisetsky DS. The role of HMGB1 in
the pathogenesis of inflammatory and autoimmune diseases. Mol Med 2014; 20: 138-146.
Shim EJ, Chun E, Lee HS, Bang BR, Kim TW,
Cho SH, Min KU, Park HW. The role of highmobility group box-1 (HMGB1) in the pathogenesis of asthma. Clin Exp Allergy 2012; 42:
958-965.
Hou C, Zhao H, Liu L, Li W, Zhou X, Lv Y, Shen
X, Liang Z, Cai S, Zou F. High mobility group
protein B1 (HMGB1) in Asthma: comparison of
patients with chronic obstructive pulmonary
disease and healthy controls. Mol Med 2011;
17: 807-815.
Nakamura T, Sato E, Fujiwara N, Kawagoe Y,
Maeda S, Yamagishi S. Increased levels of
soluble receptor for advanced glycation end
products (sRAGE) and high mobility group box
1 (HMGB1) are associated with death in patients with acute respiratory distress syndrome. Clin Biochem 2011; 44: 601-604.
Entezari M, Weiss DJ, Sitapara R, Whittaker L,
Wargo MJ, Li J, Wang H, Yang H, Sharma L,
Phan BD, Javdan M, Chavan SS, Miller EJ, Tracey KJ, Mantell LL. Inhibition of high-mobility
group box 1 protein (HMGB1) enhances bacterial clearance and protects against Pseudomonas Aeruginosa pneumonia in cystic fibrosis.
Mol Med 2012; 18: 477-485.
Wang C, Fei G, Liu Z, Li Q, Xu Z, Ren T. HMGB1
was a pivotal synergistic effecor for CpG oligonucleotide to enhance the progression of human lung cancer cells. Cancer Biol Ther 2012;
13: 727-736.
Entezari M, Javdan M, Antoine DJ, Morrow DM,
Sitapara RA, Patel V, Wang M, Sharma L, Gorasiya S, Zur M, Wu W, Li J, Yang H, Ashby CR,
Thomas D, Wang H, Mantell LL. Inhibition of
extracellular HMGB1 attenuates hyperoxia-induced inflammatory acute lung injury. Redox
Biol 2014; 2: 314-322.
Int J Clin Exp Pathol 2015;8(2):1341-1353
HMGB1 handling IL-6 and tuberculosis
[14] Rovere-Querini P, Capobianco A, Scaffidi P, Valentinis B, Catalanotti F, Giazzon M, Dumitriu IE,
Müller S, Iannacone M, Traversari C, Bianchi
ME, Manfredi AA. HMGB1 is an endogenous
immune adjuvant released by necrotic cells.
Embo Rep 2004; 5: 825-830.
[15] Saenz R, Souza CS, Huang CT, Larsson M,
Esener S, Messmer D. HMGB1-derived peptide
acts as adjuvant inducing immune responses
to peptide and protein antigen. Vaccine 2010;
28: 7556-7562.
[16] Ciucci A, Gabriele I, Percario ZA, Affabris E,
Colizzi V, Mancino G. HMGB1 and cord blood:
its role as immuno-adjuvant factor in innate
immunity. PLoS One 2011; 6: e23766.
[17] Fagone P, Shedlock DJ, Bao H, Kawalekar OU,
Yan J, Gupta D, Morrow MP, Patel A, Kobinger
GP, Muthumani K, Weiner DB. Molecular adjuvant HMGB1 enhances anti-influenza immunity during DNA vaccination. Gene Ther 2011;
18: 1070-1077.
[18] Saenz R, Futalan D, Leutenez L, Eekhout F,
Fecteau JF, Sundelius S, Sundqvist S, Larsson
M, Hayashi T, Minev B, Carson D, Esener S,
Messmer B, Messmer D. TLR4-dependent activation of dendritic cells by an HMGB1-derived
peptide adjuvant. J Transl Med 2014; 12: 211.
[19] Schierbeck H, Pullerits R, Pruunsild C, Fischer
M, Holzinger D, Laestadius Å, Sundberg E, Harris HE. HMGB1 levels are increased in patients
with juvenile idiopathic arthritis, correlate with
early onset of disease, and are independent of
disease duration. J Rheumatol 2013; 40:
1604-1613.
[20] Wu Y, Zhang K, Zhao L, Guo J, Hu X, Chen Z.
Increased serum HMGB1 is related to oxidative stress in patients with atrial fibrillation. J
Int Med Res 2013; 41: 1796-1802.
[21] Chung HW, Lee SG, Kim H, Hong DJ, Chung JB,
Stroncek D, Lim JB. Serum high mobility group
box-1 (HMGB1) is closely associated with the
clinical and pathologic features of gastric cancer. J Transl Med 2009; 7: 38.
[22] Barnay-Verdier S, Fattoum L, Borde C, Kaveri S,
Gibot S, Marechal V. Emergence of autoantibodies to HMGB1 is associated with survival in
patients with septic shock. Intensive Care Med
2011; 37: 957-962.
[23] Zhou H, Ji X, Wu Y, Xuan J, Qi Z, Shen L, Lan L,
Li Q, Yin Z, Li Z, Zhao Z. A dual-role of Gu-4 in
suppressing HMGB1 secretion and blocking
HMGB1 pro-inflammatory activity during inflammation. PLoS One 2014; 9: e89634.
[24] Sadamura-Takenaka Y, Ito T, Noma S, Oyama Y,
Yamada S, Kawahara K, Inoue H, Maruyama I.
HMGB1 promotes the development of pulmonary arterial hypertension in rats. PLoS One
2014; 9: e102482.
1351
[25] Gong G, Xiang L, Yuan L, Hu L, Wu W, Cai L, Yin
L, Dong H. Protective effect of glycyrrhizin, a
direct HMGB1 inhibitor, on focal cerebral ischemia/reperfusion-induced inflammation, oxidative stress, and apoptosis in rats. PLoS One
2014; 9: e89450.
[26] Andersson U, Tracey KJ. HMGB1 is a therapeutic target for sterile inflammation and infection.
Annu Rev Immunol 2011; 29: 139-162.
[27] Andersson U, Rauvala H. Introduction: HMGB1
in inflammation and innate immunity. J Intern
Med 2011; 270: 296-300.
[28] Musumeci D, Roviello GN, Montesarchio D. An
overview on HMGB1 inhibitors as potential
therapeutic agents in HMGB1-related pathologies. Pharmacol Ther 2014; 141: 347-357.
[29] Grover A, Troudt J, Foster C, Basaraba R, Izzo A.
High mobility group box 1 acts as an adjuvant
for tuberculosis subunit vaccines. Immunology
2014; 142: 111-23.
[30] Zeng JC, Lin DZ, Yi LL, Liu GB, Zhang H, Wan
WD, Zhang JA, Wu XJ, Xiang WY, Kong B, Chen
WZ, Wang CY, Xu JF. An essential role for BTLA
in immune memory for αβ T cells against M.
tuberculosis in patients with active pulmonary
tuberculosis. Am J Transl Res 2014; 6: 494506.
[31] Bhowmik A, Seemungal TA, Sapsford RJ,
Wedzicha JA. Relation of sputum inflammatory
markers to symptoms and lung function changes in COPD exacerbations. Thorax 2000; 55:
114-120.
[32] Huang D, Qiu L, Wang R, Lai X, Du G, Seghal P,
Shen Y, Shao L, Halliday L, Fortman J, Shen L,
Letvin NL, Chen ZW. Immune gene networks of
mycobacterial vaccine-elicited cellular responses and immunity. J Infect Dis 2007; 195:
55-69.
[33] Rowe SM, Jackson PL, Liu G, Hardison M,
Livraghi A, Solomon GM, McQuaid DB, Noerager BD, Gaggar A, Clancy JP, O’Neal W, Sorscher
EJ, Abraham E, Blalock JE. Potential role of
high-mobility group box 1 in cystic fibrosis airway disease. Am J Respir Crit Care Med 2008;
178: 822-831.
[34] Verbon A, Juffermans N, Van Deventer SJ,
Speelman P, Van Deutekom H, Van Der Poll T.
Serum concentrations of cytokines in patients
with active tuberculosis (TB) and after treatment. Clin Exp Immunol 1999; 115: 110-113.
[35] Fiorenza G, Rateni L, Farroni MA, Bogue C, Dlugovitzky DG. TNF-αlpha, TGF-beta and NO relationship in sera from tuberculosis (TB) patients
of different severity. Immunol Lett 2005; 98:
45-48.
[36] Gaini S, Koldkjaer OG, Moller HJ, Pedersen C,
Pedersen SS. A comparison of high-mobility
group-box 1 protein, lipopolysaccharide-binding protein and procalcitonin in severe commu-
Int J Clin Exp Pathol 2015;8(2):1341-1353
HMGB1 handling IL-6 and tuberculosis
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
[46]
[47]
[48]
[49]
nity-acquired infections and bacteraemia: a
prospective study. Crit Care 2007; 11: R76.
Nemeth J, Winkler HM, Boeck L, Adegnika AA,
Clement E, Mve TM, Kremsner PG, Winkler S.
Specific cytokine patterns of pulmonary tuberculosis in Central Africa. Clin Immunol 2011;
138: 50-59.
Salgame P. Host innate and Th1 responses
and the bacterial factors that control Mycobacterium tuberculosis infection. Curr Opin Immunol 2005; 17: 374-80.
Mahan CS, Zalwango S, Thiel BA, Malone LL,
Chervenak KA, Baseke J, Dobbs D, Stein CM,
Mayanja H, Joloba M, Whalen CC, Boom WH.
Innate and adaptive immune responses during
acute M. tuberculosis infection in adult household contacts in Kampala, Uganda. Am J Trop
Med Hyg 2012; 86: 690-697.
Li Y, Wang Y, Liu X. The role of airway epithelial
cells in response to mycobacteria infection.
Clin Dev Immunol 2012; 2012: 791392.
Lalvani A, Behr MA, Sridhar S. Innate immunity
to TB: a druggable balancing act. Cell 2012;
148: 389-391.
Yao S, Huang D, Chen CY, Halliday L, Wang RC,
Chen ZW. CD4+ T cells contain early extrapulmonary tuberculosis (TB) dissemination and
rapid TB progression and sustain multieffector
functions of CD8+ T and CD3- lymphocytes:
mechanisms of CD4+ T cell immunity. J Immunol 2014; 192: 2120-2132.
Veenstra H, Baumann R, Carroll NM, Lukey PT,
Kidd M, Beyers N, Bolliger CT, van Helden PD,
Walzl G. Changes in leucocyte and lymphocyte
subsets during tuberculosis treatment; prominence of CD3dimCD56+ natural killer T cells in
fast treatment responders. Clin Exp Immunol
2006; 145: 252-260.
Nitta T, Nishinakamura H, Kojima D, Mera T,
Ono J, Kodama S, Yasunami Y. HMGB1-Mediated Early Loss of Transplanted Islets Is Prevented by Anti-IL-6R Antibody in Mice. Pancreas
2015; 44: 166-171.
Gabrysova L, Howes A, Saraiva M, O’Garra A.
The regulation of IL-10 expression. Curr Top Microbiol Immunol 2014; 380: 157-90.
Gabay C. Interleukin-6 and chronic inflammation. Arthritis Res Ther 2006; 8: S3.
Korbel DS, Schneider BE, Schaible UE. Innate
immunity in tuberculosis: myths and truth. Microbes Infect 2008; 10: 995-1004.
Fraser CK, Lousberg EL, Kumar R, Hughes TP,
Diener KR, Hayball JD. Dasatinib inhibits the
secretion of TNF-αlpha following TLR stimulation in vitro and in vivo. Exp Hematol 2009; 37:
1435-1444.
Michlewska S, Dransfield I, Megson IL, Rossi
AG. Macrophage phagocytosis of apoptotic
neutrophils is critically regulated by the oppos-
1352
[50]
[51]
[52]
[53]
[54]
[55]
[56]
[57]
[58]
ing actions of pro-inflammatory and anti-inflammatory agents: key role for TNF-αlpha.
FASEB J 2009; 23: 844-854.
Ribeiro-Rodrigues R, Resende Co T, Johnson
JL, Ribeiro F, Palaci M, Sá RT, Maciel EL, Pereira
Lima FE, Dettoni V, Toossi Z, Boom WH, Dietze
R, Ellner JJ, Hirsch CS. Sputum cytokine levels
in patients with pulmonary tuberculosis as
early markers of mycobacterial clearance. Clin
Diagn Lab Immunol 2002; 9: 818-823.
Yokoyama T, Komori A, Nakamura M, Takii Y,
Kamihira T, Shimoda S, Mori T, Fujiwara S, Koyabu M, Taniguchi K, Fujioka H, Migita K, Yatsuhashi H, Ishibashi H. Human intrahepatic biliary epithelial cells function in innate immunity
by producing IL-6 and IL-8 via the TLR4-NF-kappaB and -MAPK signaling pathways. Liver Int
2006; 26: 467-476.
Lin CC, Lee IT, Yang YL, Lee CW, Kou YR, Yang
CM. Induction of COX-2/PGE(2)/IL-6 is crucial
for cigarette smoke extract-induced airway inflammation: Role of TLR4-dependent NADPH
oxidase activation. Free Radic Biol Med 2010;
48: 240-254.
Hussein MH, Kato T, Sugiura T, Daoud GA, Suzuki S, Fukuda S, Sobajima H, Kato I, Togari H.
Effect of hemoperfusion using polymyxin B-immobilized fiber on IL-6, HMGB-1, and IFN gamma in a neonatal sepsis model. Pediatr Res
2005; 58: 309-314.
Nakamura T, Sato E, Fujiwara N, Kawagoe Y,
Suzuki T, Ueda Y, Yamada S, Shoji H, Takeuchi
M, Ueda S, Matsui T, Adachi H, Okuda S, Yamagishi S. Circulating levels of advanced glycation end products (AGE) and interleukin-6 (IL-6)
are independent determinants of serum asymmetric dimethylarginine (ADMA) levels in patients with septic shock. Pharmacol Res 2009;
60: 515-518.
Norrby-Teglund A, Rouhiainen A, Rauvala H,
Herman G, Tracey KJ, Lee ML, Andersson J, Tokics L, Treutiger CJ. Persistent elevation of high
mobility group box-1 protein (HMGB1) in patients with severe sepsis and septic shock. Crit
Care Med 2005; 33: 564-573.
Gaini S, Pedersen SS, Koldkaer OG, Pedersen
C, Moestrup SK, Moller HJ. New immunological
serum markers in bacteraemia: anti-inflammatory soluble CD163, but not proinflammatory
high mobility group-box 1 protein, is related to
prognosis. Clin Exp Immunol 2008; 151: 423431.
Lantos J, Foldi V, Roth E, Weber G, Bogar L,
Csontos C. Burn trauma induces early HMGB1
release in patients: its correlation with cytokines. Shock 2010; 33: 562-7.
Choi J, Min HJ, Shin JS. Increased levels of
HMGB1 and pro-inflammatory cytokines in
children with febrile seizures. J Neuroinflammation 2011; 8: 135.
Int J Clin Exp Pathol 2015;8(2):1341-1353
HMGB1 handling IL-6 and tuberculosis
[59] Li P, Bledsoe G, Yang ZR, Fan H, Chao L, Chao
J. Human kallistatin administration reduces organ injury and improves survival in a mouse
model of polymicrobial sepsis. Immunology
2014; 142: 216-226.
[60] Miura K, Sahara H, Sekijima M, Kawai A, Waki
S, Nishimura H, Setoyama K, Clayman ES, Shimizu A, Yamada K. Protective Effect of Neutralization of the Extracellular High-Mobility Group
Box 1 on Renal Ischemia-Reperfusion Injury in
Miniature Swine. Transplantation 2014; 98:
937-43.
1353
[61] Yang R, Zou X, Tenhunen J, Zhu S, Kajander H,
Koskinen ML, Tonnessen TI. HMGB1 neutralization is associated with bacterial translocation during acetaminophen hepatotoxicity.
BMC Gastroenterol 2014; 14: 66.
[62] Cai J, Xia H, Huang Y, Lu Y, Wu Z, Jian J. Molecular cloning and characterization of high
mobility group box1 (Ls-HMGB1) from humphead snapper, Lutjanus sanguineus. Fish
Shellfish Immunol 2014; 40: 539-544.
Int J Clin Exp Pathol 2015;8(2):1341-1353