Effect of HA330 resin-directed hemoadsorption on a porcine acute respiratory distress syndrome model
Xu et al. Ann. Intensive Care
Effect of HA330 resin-directed hemoadsorption on a porcine acute respiratory distress syndrome model
Xuefeng Xu 1 2
Chune Jia 0
Sa Luo 0
Yanming Li 2 4
Fei Xiao 2 3
Huaping Dai 0
Chen Wang 0 2
0 Department of Pulmonary and Critical Care Medicine, China-Japan Friendship Hospital , 2 Yinghua Dongjie, Beijing 100029 , China
1 Department of Surgical Intensive Care Medicine, Beijing An Zhen Hospital, Capital Medical University , No. 2 Anzhen Rd., Chao-Yang District, Beijing 100029 , China
2 National Clinical Research Centre for Respiratory Diseases, Beijing Hospital , Beijing 100730 , China
3 Key Laboratory of Geriatrics, Beijing Hospital and Beijing Institute of Geriatrics, Ministry of Health , Beijing , China
4 Department of Pulmonary and Critical Care Medicine, Beijing Hospital , Beijing 100730 , China
Background: Blood purification is an emerging approach to dampening the cytokine storm. This study aims to assess the efficacy of HA330 resin-directed hemoadsorption (HA) on endotoxin-induced porcine acute respiratory distress syndrome (ARDS) model. Methods: Twenty-four Chinese domestic pigs were allocated into saline group receiving intravenous infusion of saline (N = 6) and endotoxin group receiving intravenous infusion of LPS (N = 18). When ALI model was initially diagnosed, six pigs in the LPS and saline group were killed for BALF and histopathological analysis. The remaining 12 pigs in LPS group received 3-h HA (N = 6) or HA-sham (N = 6) treatment, respectively. Following another 5-h observation, animals were killed. Variables on hemodynamics, blood gases and lung mechanics were recorded at a series of time points. Differentially expressed cytokines and proteins were determined by ELISA and proteomics. Results: HA treatment significantly improved injured oxygenation induced by LPS. HA also partially improved the barrier permeability and reduced lung edema and inflammation/injury induced by LPS infusion. Proteomic analysis showed the differentially expressed proteins between HA- and HA-sham-treated groups mostly belonged to the categories of acute inflammation/immune response, and proteolysis. Conclusions: Hemoadsorption improved ARDS possibly by blunting the cytokine storm and by restoring homeostasis of the disordered proteome milieu in the exudative phase.
ARDS; Cytokine storm; Hemoadsorption; Porcine model; Proteomics
The acute respiratory distress syndrome (ARDS) is a
condition with the acute onset of noncardiac
respiratory failure that develops in response to a series of insults
to the alveolar-capillary barrier [
]. The current
mainstay therapy for ARDS is largely supportive [
has been shown to reduce mortality by limiting further
iatrogenic injury to the already injured lungs. However,
the mortality rate of ARDS is still unacceptably high
. ARDS is largely caused by the local alveolar and
circulating “cytokine storm” that happens with bacterial/
virus infection, burn/trauma and some possible
iatrogenic factors, such as high-volume ventilation [
has encouraged the development of several specific
targeted pharmacologic therapy directed against single key
pathogenic mediators. However, most clinical trials have
proven no benefit to disease outcome and were stopped
early for futility [
]. Investigators shifted their interests
into the extracorporeal blood purification (EBP)
modality, which is a nonspecific, broad-spectrum method to
blunt the “cytokine storm” by immediately clearing a
series of circulating and resident mediators [
Until now, there are only some inconsistent preliminary
data to investigate the effects of hemofiltration (a
modality of EBP) on ARDS animal models [
]. Also, it has
been shown that hemoadsorption (HA), another
modality of EBP, can improve oxygenation in septic patients
]. However, evidences of HA’s effects on ARDS
patients animal models are scarce. We hypothesize that
HA can improve oxygenation, reduce the permeability of
alveolar-capillary barrier and alleviate pulmonary edema/
damage by blunting the circulating/alveolar cytokines in
the process of ARDS. To test this hypothesis, we
established an endotoxin-induced ARDS porcine model to
explore HA’s therapeutic effect by using a HA330 resin
cartridge (a new weapon against cytokine storm).
Research animals and ethical considerations
This study was approved by the Institutional Animal
Experiment Committee of Beijing Hospital. All
experiments were performed according to the Declaration of
Helsinki conventions for the use and care of animals.
Twenty-four healthy, 3–4-week-old Chinese domestic
white pigs of both sexes were chosen from a local stock
routinely used for the experimental research. The
average weight of pigs used in this experimental procedure is
43 ± 0.3 kg (mean ± SEM).
Sedation, anesthesia and muscle relaxation
Detailed information is shown in Additional file 1:
Ventilation and measurements of lung mechanics
Animals were ventilated in a volume-controlled mode.
The ventilation protocol at baseline used a tidal volume
of 10 ml/kg body weight, a fraction of inspired
oxygen (FiO2) of 0.4 and a positive end-expiratory pressure
(PEEP) at 0 cmH2O. Parameters of lung mechanics were
recorded. Detailed information is shown in Additional
file 1: Supplement-Methods.
Instrumentation and hemodynamic measurements
Cardiac output (CO), mean arterial pressure (MAP),
systematic vascular resistance (SVR), mean pulmonary
arterial pressure (MPAP), pulmonary vascular
resistance (PVR), pulmonary artery wedge pressure (PAWP),
extravascular lung water (EVLW) and pulmonary
vascular permeability index (PVPI) were measured with
PiCCO and Swan-Ganz systems. Detailed information is
shown in Additional file 1: Supplement-Methods.
After surgical preparations and instrumentation, all
animals were ventilated in prone position and a period of 1 h
was left for animal stabilization. Thereafter, baseline (i.e.,
the time point after 1-h stabilization) parameters
including lung mechanics, hemodynamics, blood gases were
recorded and venous blood samples were collected. As a
next step, lung injury model was produced with i.v.
infusion of 50 μg/kg LPS (Escherichia coli, serotype O55:B5,
Sigma-Aldrich, St. Louis, MO, USA) mixed in 500 ml of
saline that was delivered via a volumetric infusion pump
(Instrumentation Laboratory, Bedford, MA, USA) for
about 2 h [
]. Before the start of the experiment, 24
animals were purchased and randomly divided into four
groups according to different conditions: (a) control group
(N = 6), animals received an i.v. infusion of saline without
LPS (LPS-sham); (b) LPS group (N = 6), animals received
an i.v. infusion of saline with LPS as indicated above. When
experimental ARDS was diagnosed in LPS group (PaO2/
FiO2 is equal or less than 200 mmHg with a PEEP equal to
5 cmH2O), animals in control and LPS group were all killed
for bronchoalveolar lavage fluid (BALF) and histologic
assessment in order to aid further diagnosis of lung injury.
(c) LPS plus HA-sham group (N = 6), animals received an
i.v. infusion of saline with LPS. Immediately after ARDS was
well established, HA-sham treatment was started by using
a hollow fiber membrane hemofilter (Fresenius) with the
ultrafiltration line clamped. Two animals in this group were
excluded because they died or developed severe
sepsis/septic shock and refractory acidosis despite active liquid
recovery and vasopressor administration before moderate ARDS
diagnosis; (d) LPS plus HA group (N = 6), animals received
an i.v. infusion of saline with LPS, and real HA treatment
was started by using a disposable hemoperfusion cartridge
(HA330 resin, styrene divinylbenzene copolymers, with a
blood flow of 200–250 ml/min, volume of 185 ml; Jafron
Biomedical Co., Ltd., Zhuhai, GuangDong, China; http://
www.jafron.com/). Two pigs were also discarded because
they died of septic shock and severe disturbance of water,
electrolyte and acid base before the establishment of
hemoperfusion circulation. HA-sham and HA treatment
lasted for 3 h, and thereafter, the animals were monitored
for an extended observation period of 5 h and then killed
with a bolus injection of 10 ml of 15% KCl. BAL fluid
collection and lung tissue harvesting were performed
immediately upon exsanguination and sternotomy performance
for the measurements of inflammatory markers, proteomes
and for histologic evaluation. Parameters of
hemodynamics, blood gases and lung mechanics were recorded every
hour following the start of LPS or sham LPS infusion until
the end of experiment. Plasma samples were also prepared
in the corresponding time points. During the experiment,
all pigs were continuously infused with Ringer lactate
solution or saline at a rate of 12 ml/kg/h at the start of the
experiment. The infusion rate was increased to 15–20 ml/
kg/h if the MAP was less than 70–50 mmHg. However, if
pulmonary artery wedge pressure (PAWP) continuously
raised and exceeded 12 mmHg, the rate of infusion should
be reduced to prevent fluid overload. Meanwhile,
norepinephrine (0.5–1.5 μg/kg/min) was then used to achieve
this level of mPAP in LPS-treated pigs. Body temperature
was maintained over 36 °C by using a heating blanket. The
experimental protocol was summarized in Additional file 2:
Assessment of the LPS‑induced ARDS porcine model
To determine whether ARDS has occurred, at least four
main features of ARDS should be assessed: (1)
abnormalities of gas exchange. A ratio of the partial pressure of
arterial oxygen to the fraction of inspired oxygen (PaO2/
FiO2) ≤ 200 mmHg at PEEP = 5 cmH2O (clinical
diagnostic criteria of moderate ARDS) is the initial
evaluating criteria;(2)a remarkable leakage of alveolar barrier as
indicated by the significant increase of lung wet-to-dry
weight ratio, EWLW and PVPI; (3) a significant
infiltration of inflammatory cells and production of
inflammatory cytokines in both circulation and lung tissues (plasma
and lung IL-6, IL-8, IL-1β, IL-17A and TNF-α levels were
measured in our study); (4) diffuse alveolar damage (DAD)
as determined by lung histopathological examination [
HA330 cartridge‑directed hemoadsorption
The HA330 is an electrically neutral microporous
resin that is a powerful new weapon in the
clearance of “cytokine storm” occurred in sepsis [
Detailed information is shown in Additional file 1:
Sixty milliliter ice-cold saline was used to collect BALF in
the left upper, middle and lower lobes. Detailed
information is shown in Additional file 1: Supplement-Methods.
Assessment of total protein contents in BALF
BCA (bicinchoninic acid) protein assay reagent kit
(Pierce Biotechnology, Rockford, IL, USA) was used.
Detailed information is shown in Additional file 1:
Lung tissue collection and histopathological evaluation
The right lower lobe was excised, fixed and sliced for
routine hematoxylin and eosin (H&E) staining. ALI
scoring was done according to a consensus report
published previously [
] (Additional file 3: Table S1).
Detailed information is shown in Additional file 1:
Lung water assessed by gravimetric method
Samples from unused right lung lobes were dissected
free of non-lung parenchymal tissue, placed in a dish and
weighed (wet weight). Then, they were dried in an oven
at 80 °C and weighed daily until their weight was
maintained unchanged (dry weight). The total water content
of the lung was crudely estimated as a wet-to-dry weight
Concentrations of proinflammatory mediators in circulation and lung tissues
IL-1β, IL-6, IL-8, TNF-α and IL-17A were measured
by ELISA kits (Bluegene Biotech CO., LTD, Shanghai,
China). Detailed information is shown in Additional
file 1: Supplement-Methods.
Plasma and lung proteome
Protein samples of plasma, BALF and lung tissue in HA
or HA-sham treatment group were collected and received
iTRAQ-labeled (Applied Biosystems, Foster City, CA,
USA) mass spectrometric analysis. Bioinformatics
analysis was also performed [
]. Detailed information is
shown in Additional file 1: Supplement-Methods.
The data are shown as the mean ± SEM. Parametric data
between multiple groups were compared by using the
Kruskal–Wallis test and followed by Mann–Whitney U
test if statistically significant. Mann–Whitney U test was
also used for the comparison between two independent
continuous variables. Data were analyzed using the SPSS
statistical software package for Windows, version 13.0
(SPSS, Chicago, IL, USA), and P < 0.05 was considered as
Intravenous infusion of endotoxin causes sharp changes in physiological parameters
The ratio of PaO2/FiO2 and alveolar-arterial oxygen
partial pressure difference (AaDO2) in pigs that received
LPS infusion declined gradually (from 440 ± 19 to
157 ± 23 mmHg, P < 0.001, Fig. 1a; Table 1). After LPS
infusion, CO was firstly increased and then decreased
when moderate ARDS was diagnosed. However, the
changes did not reach statistical difference when
compared with saline group. LPS infusion increased MPAP
(18.8 ± 1.5 vs. 41.0 ± 1.7 mmHg, P < 0.001, Fig. 1b;
Table 2) and PVRI (201 ± 13 vs. 781 ± 74 dyn s/cm5/m ,
P < 0.001, Table 2). However, MAP and SVRI continued
to decrease (Table 2). In addition, pulmonary mechanics
were also damaged as shown by an increase in PAWPeak,
PAWPlateau and resistance and a decrease in pulmonary
static compliance (Fig. 1c, d; Table 1).
Intravenous infusion of endotoxin causes high‑permeability pulmonary edema and lung histologic damages
The EVLWI continuously increased after LPS infusion
until ARDS was diagnosed (from 8.0 ± 0.5 ml/kg in
baseline to 15.4 ± 1.3 ml/kg at the end of this experiment
when ARDS was established, P < 0.001; Fig. 1e). In
parallel, there was also a remarkable increase in lung wet/
dry ratio in LPS-challenged pigs compared with saline
group (Fig. 1h). The alveolar-capillary membrane was
also seriously injured by LPS infusion as manifested by a
significant increase in both pulmonary vascular
permeability index (PVPI) and whole BAL protein concentration
(Fig. 1f, g). Infusion of LPS in the endotoxin challenge
group was also associated with an increase in the
numbers of whole BAL cell counting (Fig. 1i). To determine
the histopathological features of ALI model, subset of
animals (N = 6 in LPS and saline group, respectively)
were killed when ARDS model was diagnosed based
on oxygenation parameter (PaO2/FiO2 ≤ 200 mmHg at
PEEP = 5 cmH2O). Histologic examinations from
animals receiving LPS demonstrated alveolar bleeding,
microatelectasis, perivascular edema, marked leukocyte
sequestration in alveolar septa and lumen, thickened
alveolar walls, and the presence of proteinaceous debris
in the alveolar space as compared to control subjects
(Fig. 1j–l, Additional file 4: Fig. S2).
Intravenous infusion of endotoxin augments the expressions of systemic and pulmonary inflammatory cytokines
As shown by ELSA assay in Fig. 2, there was no
difference in the baseline concentration of plasma IL-1β
(Fig. 2a), IL-6 (Fig. 2d), IL-8 (Fig. 2g), TNF-α (Fig. 2j)
and IL-17A (Fig. 2m) between LPS and saline groups,
but all of these cytokines increased rapidly when
challenged with LPS infusion at T0 compared with saline
infusion. We also determined the levels of these
inflammatory cytokines within BALF and lung homogenates
479 ± 34
157 ± 23***
31.8 ± 1.49
44.7 ± 5.0**
60 ± 4
224 ± 48**
17.3 ± 1.8
35.7 ± 2.5***
13.8 ± 2.2
27.3 ± 3.2***
8.1 ± 1.3
14.7 ± 2.2
32.8 ± 3.3
16.7 ± 1.8**
4.2 ± 0.4
3.4 ± 0.3
102 ± 8
54 ± 5***
1909 ± 218
1160 ± 97*
18.5 ± 1.2
41.0 ± 1.7***
201 ± 13
781 ± 74***
6.8 ± 1.2
12.2 ± 1.4*
10.0 ± 0.9
15.4 ± 1.3***
2.9 ± 0.2
5.6 ± 0.5***
Oxygenation and ventilation parameters in saline control and LPS challenge animals at baseline and a series of time points until the end of the experiment when ALI
model was diagnosed (data shown are mean ± SEM, N = 6 of each group; *P < 0.05; **P < 0.01; ***P < 0.001 versus control group). PaO2, oxygen partial pressure; FiO2,
inspiratory oxygen fraction; PaCO2, carbon dioxide partial pressure; PAWPeak, peak airway pressure; PAWPlateau, plateau airway pressure
Hemodynamic parameters are compared between the saline control and LPS-challenged group. (Data are mean ± SEM, N = 6 of each group; *P < 0.05, **P < 0.01,
***P < 0.001; versus control group)
CO cardiac output, MAP mean arterial pressure, SVRI systemic vascular resistance index, MPAP mean pulmonary arterial pressure, PVRI pulmonary vascular resistance
index, PAWP pulmonary artery wedge pressure, EVLWI extravascular lung water index, PVPI pulmonary vascular permeability index
to assess the inflammatory response in the alveolar
compartment. ELISA assay showed significant increased
levels of these cytokines in BALF and lung homogenates
after LPS challenge except IL-17A, which was
remarkable increased in BALF but remained unchanged in lung
homogenates at T0 (Fig. 2).
Hemoadsorption improves oxygenation and lung mechanics in pigs challenged with endotoxin
We found that treatment with HA increased
LPSimpaired oxygenation (387 ± 10 mmHg at baseline vs.
183 ± 6 mmHg at T0 vs 315 ± 22 mmHg at 8 h after HA;
Fig. 3a; Table 3). In contrast, in endotoxin-challenged
pigs that did not receive HA (HA-sham) treatment, the
ratio of PaO2/FiO2 was only slightly increased at 8 h after
HA-sham (Fig. 3a; Table 3). Treatment with HA
significantly or showed a trend toward decreasing PAWPeak,
PAWPlateau, lung resistance and the impairment of static
compliance induced by endotoxin infusion when all pigs
data were pooled together and compared with HA-sham
group (Fig. 3c, d; Table 3). However, HA did not alter
the impairment of systemic/pulmonary hemodynamic
parameters (Fig. 3b; Table 4).
Hemoadsorption blunts lung edema and histopathological signs of ARDS
Compared with LPS + HA-sham group, HA therapy
for ARDS pigs caused a decrease in EVLWI, PVPI, lung
wet-to-dry weight ratio and BALF cell count (Fig. 3e, f,
h, i; Table 4). However, the changes did not reach
statistical difference when compared with LPS +
HAsham group. But HA therapy can significantly reduce
total BALF protein as compared to HA-sham
treatment (Fig. 3g). These beneficial alterations were further
reflected by reduced histologic signs of inflammation
and injury (Fig. 3j–l).
Hemoadsorption reduces circulating and alveolar cytokine levels
ELISA assay showed that plasma level of IL-1β and IL-6
induced by endotoxin was significantly blunted by HA
treatment compared with HA-sham treatment (Fig. 4a,
d). HA also elicited a remarkable decrease in the
expression of IL-1β and IL-6 in both BALF and lung
homogenate (Fig. 4b, c, e, f ). Plasma and lung homogenate levels
of IL-8 were also decreased (Fig. 4g, i). BALF levels of
TNF-α and IL-17A were significantly reduced by HA
treatment (Fig. 4k, n).
Hemoadsorption‑altered plasma and pulmonary proteome
First, plasma proteome was analyzed. The differentially
accumulated proteins (T0 vs baseline) and their dynamic
changes after HA/HA-sham treatment (8 h after T0 vs.
baseline) are shown in Additional file 5: Fig. S3. These
proteins were classified into several categories based on
biological process analysis (Tables 5, 6). We identified four
Oxygenation and ventilation parameters in LPS + HA (sham)- and LPS + HA-treated animals at baseline and a series of time points until the end of the experiment
(data are mean ± SEM, N = 3–4 of each group; *P < 0.05 when compared between groups; †P < 0.05 when compared with baseline within per group; §P < 0.05
versus T0 within per group). PaO2, oxygen partial pressure; FiO2, inspiratory oxygen fraction; PaCO2, carbon dioxide partial pressure; PAWPeak, peak airway pressure;
PAWPlateau, plateau airway pressure
Table 4 Systemic and pulmonary hemodynamic measurements compared between LPS + HA (sham) and LPS + HA
Hemodynamic parameters are shown in the LPS + HA (sham) and LPS + HA groups. Data are mean ± SEM, N = 3–4 of each group; CO, cardiac output; MAP, mean
arterial pressure; SVRI, systemic vascular resistance index; MPAP, mean pulmonary arterial pressure; PVRI, pulmonary vascular resistance index; PAWP, pulmonary artery
wedge pressure; EVLWI, extravascular lung water index; PVPI, pulmonary vascular permeability index
plasma proteins, i.e., interleukin-1 receptor antagonist
protein (IL-1Ra), inter-alpha-trypsin inhibitor heavy chain
H4 (ITIH4), matrix metallopeptidase (MMP)-1 and
MMP10, that were up-regulated at T0 in these two groups,
but sharply reduced after HA treatment while further
increased in HA-sham group (Fig. 5a, b). Differentially
expressed proteins (these proteins were comparable at T0
when compared with HA with HA-sham group) after 8-h
HA or HA-sham treatment, and their GO annotations are
also shown in Additional file 6: Table S2 and Additional
file 7: Fig. S4. We then analyzed proteome in BALF and
lung homogenate samples. Differentially expressed
proteins compared between HA and HA-sham groups are
shown in Additional file 8: Table S3, Additional file 9: Table
S4, Additional file 10: Table S5, Additional file 11: Table
S6. The most five relevant GO categories (P value less
than 0.05) are shown in Fig. 5c, d. KEGG pathway
analysis showed that these differentially expressed proteins were
involved in the canonical signaling pathways (Fig. 5e, f ).
In this current study, we tested the efficacy of “sorbent
strategy”-based HA on a porcine ARDS model and found
that HA reduced circulating and alveolar levels of
proinflammatory cytokines, improved oxygenation and
attenuated lung injuries in the exudative phase. This provides
some clue that HA330 cartridge may be a novel potential
weapon fighting against the “cytokine storm” on the
alveolar-capillary membrane barrier.
The most commonly used large-animal models of ARDS
include endotoxin infusion, repeated lavage, oleic acid and
smoke/burn injury [
]. To reproduce the most known
risk factor and etiology for ARDS, which is sepsis [
systemically administrated endotoxin (LPS) to mimic the
clinically relevant sepsis-induced ARDS. The
susceptibility to LPS is highly variable and differs among different
animals. Pigs, sheep, calves, and cats are more sensitive
to LPS challenge. Low dosage of LPS (μg/kg range) can
induce significant ARDS-like features in these animals. In
contrast, animals such as rodents or dogs require much
higher doses to develop lung injury (mg/kg range). This
different vulnerability to LPS may be induced by the
pulmonary intravascular macrophages (PIM), a lung
resident population of mature macrophage [
]. 8–100 μg/
kg of LPS has been used in several previous studies to
develop porcine ARDS model [
12, 20, 21
]. In our study,
low amount of LPS (50ug/kg in 500 ml saline for 2 h) can
also induce the pigs to develop an abrupt decrease in
oxygenation and arterial pressure and a decrease in systemic
vascular resistance, needing rapid fluid resuscitation (10–
20 ml/kg/h of saline) and the usage of vasopressor
infusion (0.5–1.5 μg/kg/min of norepinephrine).
In most recent large-animal studies, the diagnosis of
experimental ARDS is ambiguous without definite
12, 18, 21, 22
]. To determine whether ARDS really
occurred, we adopt the four diagnostic criteria released
by ATS official statement on the definition of ARDS
in animals . ARDS model was initially diagnosed
when PaO2/FiO2 is less than 200 mmHg with a PEEP at
5 cmH2O (criteria (a) moderate ARDS in human beings
according to Berlin definition). Then, the ARDS model
was further assessed with other criteria listed as follows:
(b) permeability. Permeability assessment was performed
by using four parameters, i.e., EVLWI, PVPI, BALF
protein and the ratio of wet to dry lung weight (W/D ratio).
EVLW is the amount of interstitial and alveolar fluid
]. Previous studies showed that EVLW indexed to
body weight (EVLWI) is well correlated with the value
obtained by gravimetry and can independently predict
mortality in septic and ARDS patients [
]. We found that
both EVLWI and W/D ratio were significantly increased
after LPS infusion, indicating an exudative edema phase
(Fig. 1; Table 2). The permeability of lung epithelium
barrier can also be calculated by PVPI [
]. PVPI in this
model was increased to 5.6 ± 0.5 at T0 when ARDS was
diagnosed (Fig. 1; Table 2), indicating high permeable
(rather than hydrostatic) pulmonary edema because it
was reported that a PVPI value of 3 can distinguish these
two forms of pulmonary edema [
]. Accordingly, BALF
protein concentration, another leakage marker [
also increased after LPS infusion (Fig. 1g). (c) Histology.
To our knowledge, this is the first large-animal study to
assess histologic changes at the time point that ARDS was
initially diagnosed by oxygenation and ahead of therapy
implementation. The major histologic findings are shown
in Fig. 1j–l. It is noteworthy that LPS i.v. infusion causes
only modest lung edema (perivascular edema, Additional
file 4: Fig. S2) as compared to ARDS patients, because
the endothelium barrier is more resistant to the damage
induced by LPS [
]. (d) Inflammation. We found that
both circulating and alveolar inflammatory cytokines
were elevated, indicating an acute “cytokine storm” was
elicited in systemic and lung milieu by i.v. infusion of LPS.
Thereafter, we conducted subsequent studies to explore
the “therapeutic,” rather than “prophylactic” effect of
hemoadsorption (HA) on the well-established (or
fardeveloped, rather than “immature”) ARDS model. HA
was performed with the HA330 neutral microporous
resin cartridge specially designed for the absorption
of medium-sized inflammatory cytokines. First, we
found that HA treatment increased PaO2/FiO2
gradually (Fig. 3). Several previous studies in septic patients
attributed this beneficial effect to the improved
]. However, a previous experimental
study showed that zero-balanced high-volume
continuous veno-venous hemofiltration (CVVH) could improve
arterial oxygenation without systemic or pulmonary
hemodynamics improvement . We also found that
animals treated with or without HA330-based
hemadsorption did not differ in their levels of MAP, SVRI, MPAP
and PVRI (Table 4). Considering that HA therapy
elicited a trend in reducing lung water and the permeability
of alveolar-capillary membrane barrier (Fig. 3e–i), we
assume that oxygenation improvement should be partly
due to the reduction of alveolar fluid leakage after
clearing the peak concentration of alveolar cytokines,
corresponding with the “peak concentration hypothesis”
for EBP modality [
]. Lung local cytokines removal
may be the result of passive spillover or active transport
]. Notably, a recent clinical study showed that the
a Note that changes are expressed as relative abundance of the plasma proteins at T0 or 8 h after HA-sham treatment compared with baseline within the LPS + HA
(sham) group. A fold change ≥ 1.20, p < 0.05 represents more protein abundance in T0 or 8 h versus baseline. By contrast, a fold change ≤ 0.83, p < 0.05 represents
less protein abundance in T0 or 8 h versus baseline
circulating and BALF levels of IL-17A were significantly
increased in ARDS patients and predicted an increased
influx of alveolar neutrophils, alveolar permeability and
organ dysfunction [
]. Thus, reduced BALF level of
IL17A in HA treatment group may be one of the reasons
for fewer lung inflammatory cells and decreased acute
lung injury. Taken together, by direct/indirect removal of
a variety of pathogenic proinflammatory mediators that
over-expressed in plasma and lung, HA timely blunted
the “cytokine storm” in the process of ARDS and restored
immunologic balance at a much lower set-point. These
findings were in line with a recent small clinical study on
septic-induced ARDS patients [
In the last step, we also examined whether HA can
effect the whole proteomes in plasma and lung, which
has not been previously reported. We found four plasma
proteins that were most differentially regulated by HA
and HA-sham therapy, i.e., IL-1Ra, MMP-1, MMP-10 and
ITIH4 (Fig. 5a, b). Soluble IL-1Ra, an important member
in IL-1 family of cytokines [
], was dramatically elevated
in ARDS and septic patients [
]. A persistent elevated
level of IL-1Ra indicates immunoparalysis, which greatly
contributes to the later deaths who survive the initial
cytokine storm in ARDS . ITIH4 is a plasma
glycoprotein that belongs to a serine protease inhibitor family
and acts as an acute-phase protein in several diseases [
However, there is a lack of studies aiming to determine the
role of ITIH4 as a potential diagnostic or prognostic
indicator for ARDS. The matrix metalloproteinases (MMPs)
are a family of proteolytic enzymes with the capacity of
degrading the extracellular matrix component, thus
causing tissue damage in the pathological process [
Previous studies were largely focused on the BAL fluid level
of MMPs (including MMP2, MMP8 and MMP9) and
found an elevated expression pattern [
]. However, the
circulating and BALF levels of MMP-1 (interstitial
collagenase) and MMP-10 (Stromeolysin 2) in ARDS animals
or patients are remained largely unknown. Collectively,
we concluded a composite of plasma biomarkers with
IL-1Ra, MMPs and ITIH4 may be useful to predict the
severity of ARDS. Of course, this hypothesis needs further
a Note that changes are expressed as relative abundance of the plasma proteins at T0 or 8 h after HA treatment compared with baseline within the LPS + HA group.
A fold change ≥1.20, P < 0.05 represents more protein abundance in T0 or 8 h versus baseline. By contrast, a fold change ≤0.83, P < 0.05 represents less protein
abundance in T0 or 8 h versus baseline
We also determined the role of HA330
cartridgebased HA on BALF and lung homogenate proteome
(Additional file 8: Table S3, Additional file 9: Table S4,
Additional file 10: Table S5, Additional file 11: Table S6).
Notably, we found that the BALF level of secreted
histone H2A, H2B and H4 was significantly blunted after
HA therapy compared with HA-sham-treated pigs.
Extracellular histones are the constituent of
neutrophil extracellular traps (NETs) structures that ensnare
and kill bacteria [
]. Elevated extracellular histones
in BALF samples from humans with ARDS have been
]. Also, instillation of neutralizing
antihistone H2A/H4 antibody reduced experimental ARDS
]. Because extracellular histones origin from
actively secretion by activated inflammatory cells, or
from passively release by necrotic cells, we concluded
that HA can significantly reduced the BALF level of
histones by attenuating inflammatory lung injuries.
BALF and lung level of Surfactant protein (SP)-B and
SP-C were also significantly up-regulated by HA
versus HA-sham treatment. Previous studies showed
decreased levels of SP-A, SP-B and SP-C in BALF of
ARDS patients [
]. Thus, HA treatment may improve
oxygenation by restoring the alveolar levels of
surfactant proteins. Collectively, we showed with the first
experience that HA330-directed HA performance has
a profound impact on plasma and lung proteome in a
sepsis-induced ARDS porcine model.
Our studies have some strength. First, we
comprehensively assessed the lung histologic features and other
biomarkers when the ARDS model was initially
diagnosed by oxygenation. Second, we first assessed the
effect of HA on experimental ARDS model by using
iTRAQ-labeled proteomic technology. However, our
studies also have some limitations. This is a small
sample animal study. Also, the effect of HA330 directed HA
should be further tested by other ARDS models. Third,
the experimental data cannot be directly introduced into
the real clinical practice. There is still a long way to go
that HA-330 cartridge-based HA can be used in ARDS
In conclusion, HA330 resin-based HA attenuated
experimental ARDS by blunting circulating and lung
“cytokine storm,” improving permeability of alveolar
barrier and promoting the recovery of the disordered
Additional file 1. Supplement-Methods.
Additional file 2: Fig. S1. Experimental protocol. Pigs received
surgical preparation for instrumentation and divided into 4 groups. ALI was
induced by intravenously infusion of LPS infusion (50 μg/kg over 2 h
dissolved in 500 ml saline). Saline control group were challenged by
equal amount of saline instead of LPS. When ALI was initially diagnosed
(PaO2/FiO2 ≤ 200 mmHg with PEEP = 5 cmH2O; the time point was set
as T0), pigs in LPS and saline groups were sacrificed for BAL and histology
examination in order to further assess the ALI model. Other subset of pigs
were subsequently treated with either HA-sham (LPS+HA-sham group),
or HA (LPS+HA group) performance 3 h. Following this, the observation
period was 5 h until the end of the experiment. Pigs in the 2 groups were
sacrificed for BAL and histology examination. Physiological variables were
recorded as indicated.
Additional file 3: Table S1. Lung injury scoring system.
Additional file 4: Fig. S2. Typical lung pathological changes induced
by i.v. infusion of LPS. Representative pig lung sections stained with HE
are shown. (A): Note atelectasis and the thickened alveolar walls. The
majority of the alveoli are infiltrated with inflammatory cells (40×). (B):
Arrow shows patchy neutrophilic infiltrates in alveolar spaces; “†”indicates
thickened alveolar walls with septal neutrophils (400×). (C): Note the
presence of deposition of pink fibrin strands (asterisk) and septal neutrophils
(400×). (D): Note the perivascular edema with interstitial neutrophilic
Additional file 5: Fig. S3. Dynamic expressions of plasma proteins
showed with hierarchical cluster analysis. The heatmap represents the log2
transformed fold change for each protein indicated. Columns represent
comparisons between T0/baseline and 8 h after T0/baseline in HA and
HA-sham treatment group, respectively; rows represent protein accession
numbers. Red colors indicate up-regulated proteins and green colors
indicate down-regulated proteins, respectively.
Additional file 6: Table S2. Ontology groups and associated
differentially expressed plasma proteins in the time points of 8 h after HA versus
Additional file 7: Fig. S4. Hierarchical clustering of differentially
accumulated proteins compared between HA and HA-sham treatment groups.
The heatmap represents the log2 transformed fold change for each
protein indicated. Columns represent comparisons between HA and
HAsham treatment groups at T0 and 8 h after T0, respectively; rows represent
protein accession numbers. Red colors indicate up-regulated proteins and
green colors indicate down-regulated proteins, respectively.
Additional file 8: Table S3. BALF proteins with significantly lower
expression in LPS + HA versus LPS+HA (sham)-treated pigs.
Additional file 9: Table S4. BALF proteins with significantly higher
expression in LPS + HA- versus LPS+HA (sham)-treated pigs.
Additional file 10: Table S5. Lung homogenate proteins with
significantly lower expression in LPS+HA versus LPS + HA (sham)-treated pigs.
Additional file 11: Table S6. Lung homogenate proteins with
significantly higher expression in LPS + HA versus LPS + HA (sham)-treated
AaDO2: Alveolar–arterial oxygen partial pressure difference; ARDS: Acute
respiratory distress syndrome; CO: Cardiac output; CVVH: Continuous
venovenous hemofiltration; DAD: Diffuse alveolar damage; EBP: Extracorporeal
blood purification; EVLW: Extravascular lung water; HA: Hemadsorption; H4,
ITIH4: Inter-alpha-trypsin inhibitor heavy chain; IL-1Ra: Interleukin-1 receptor
antagonist protein; PEEP: Positive end-expiratory pressure; MMP: Matrix
metallopeptidase; MAP: Mean arterial pressure; MPAP: Mean pulmonary arterial
pressure; NETs: Neutrophil extracellular traps; PVPI: Pulmonary vascular
permeability index; PIM: Pulmonary intravascular macrophages; PVRI: Pulmonary
vascular resistance index; SVRI: Systemic venous resistance index.
XX designed, made, analyzed the experiments and drafted the manuscript. CJ
designed and made the experiments. L Sa participated in the design and
performance of the experiments. F Xiao, Y Li and H Dai participated in the analysis
of the data and the critical revision of the article. C Wang contributed to the
conception and design of the study, the analysis and interpretation of the
data, revision of the article and final approval of the version to be published.
All authors read and approved the final manuscript.
We would like to thank Dr. Mingxu Shang, Dr. Hangyong He, Dr. Xiao Tang and
Dr. Bing Sun, Department of Respiratory and Critical Care Medicine, Beijing
Chao-Yang Hospital for their for their excellent technical assistance.
The authors declare that they have no competing interests.
Availability of supporting data
All the data are available and will be submitted if required.
Consent for publication
All the coauthors approve the publication of this manuscript.
Ethical approval and consent to participate
This study was approved by the Institutional Animal Experiment Committee of
This work was supported by Grants from National Natural Science Foundation
of China (Nos. 81490534, 81490530, 81430001).
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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