- Access to this full-text is provided by Springer Nature.
- Learn more
Download available
Content available from Journal of Inflammation
This content is subject to copyright. Terms and conditions apply.
S H O R T R E P O R T Open Access
Serp-2, a virus-derived apoptosis and
inflammasome inhibitor, attenuates liver
ischemia-reperfusion injury in mice
Jordan R. Yaron
1†
, Hao Chen
2†
, Sriram Ambadapadi
1†
, Liqiang Zhang
1
, Amanda M. Tafoya
1
, Barbara H. Munk
1
,
Dara N. Wakefield
3
, Jorge Fuentes
4
, Bruno J. Marques
4
, Krishna Harripersaud
4
, Mee Yong Bartee
4
,
Jennifer A. Davids
4
, Donghang Zheng
5
, Kenneth Rand
3
, Lisa Dixon
3
, Richard W. Moyer
5
, William L. Clapp
3
and
Alexandra R. Lucas
1,4,5*
Abstract
Background: Ischemia-reperfusion injury (IRI) is an antigen-independent, innate immune response to arterial occlusion
and ischemia with subsequent paradoxical exacerbation after reperfusion. IRI remains a critical problem after vessel
occlusion and infarction or during harvest and surgery in transplants. After transplant, liver IRI (LIRI) contributes to
increased acute and chronic rejection and graft loss. Tissue loss during LIRI has been attributed to local macrophage
activation and invasion with excessive inflammation together with hepatocyte apoptosis and necrosis. Inflammatory
and apoptotic signaling are key targets for reducing post-ischemic liver injury.
Myxomavirus is a rabbit-specific leporipoxvirus that encodes a suite of immune suppressing proteins, often with
extensive function in other mammalian species. Serp-2 is a cross-class serine protease inhibitor (serpin) which inhibits
the inflammasome effector protease caspase-1 as well as the apoptotic proteases granzyme B and caspases 8 and 10.
In prior work, Serp-2 reduced inflammatory cell invasion after angioplasty injury and after aortic transplantation in
rodents. In this report, we explore the potential for therapeutic treatment with Serp-2 in a mouse model of LIRI.
Methods: Wildtype (C57BL/6J) mice were subjected to warm, partial (70%) hepatic ischemia for 90 min followed by
treatment with saline or Serp-2 or M-T7, 100 ng/g/day given by intraperitoneal injection on alternate days for 5 days.
M-T7 is a Myxomavirus-derived inhibitor of chemokine-GAG interactions and was used in this study for comparative
analysis of an unrelated viral protein with an alternative immunomodulating mechanism of action. Survival, serum ALT
levels and histopathology were assessed 24 h and 10 days post-LIRI.
Results: Serp-2 treatment significantly improved survival to 85.7% percent versus saline-treated wildtype mice (p=
0.0135), while M-T7 treatment did not significantly improve survival (p= 0.2584). Liver viability was preserved by Serp-2
treatment with a significant reduction in serum ALT levels (p= 0.0343) and infarct scar thickness (p= 0.0016), but with
no significant improvement with M-T7 treatment. Suzuki scoring by pathologists blinded with respect to treatment
group indicated that Serp-2 significantly reduced hepatocyte necrosis (p=0.0057) and improved overall pathology
score (p= 0.0046) compared to saline. Immunohistochemistry revealed that Serp-2 treatment reduced macrophage
infiltration into the infarcted liver tissue (p= 0.0197).
(Continued on next page)
© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
* Correspondence: arlucas5@asu.edu
Jordan R. Yaron, Hao Chen, Sriram Ambadapadi, are Co-first authors
1
Center for Personalized Diagnostics and Center for Immunotherapy, Vaccines and
Virotherapy, Biodesign Institute, Arizona State University, Tempe, AZ, USA
4
Divisions of Cardiovascular Medicine and Rheumatology, Department of
Medicine, University of Florida, Gainesville, FL, USA
Full list of author information is available at the end of the article
Yaron et al. Journal of Inflammation (2019) 16:12
https://doi.org/10.1186/s12950-019-0215-1
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
(Continued from previous page)
Conclusions: Treatment with Serp-2, a virus-derived inflammasome and apoptotic pathway inhibitor, improves survival
after liver ischemia-reperfusion injury in mouse models. Treatment with a cross-class immune modulator provides a
promising new approach designed to reduce ischemia-reperfusion injury, improving survival and reducing chronic
transplant damage.
Keywords: Ischemia-reperfusion injury, Liver, Serpin, Immune modulation, Inflammation, Necrosis
Introduction
Ischemia-reperfusion injury (IRI) is a two-step process char-
acterized by an initial transient blockade of blood flow and
oxygen delivery. With IRI, there is an initial, sub-lethal dam-
age followed by restoration of blood flow and a paradoxical
acceleration of injury. Induction of liver ischemia-reperfusion
injury (LIRI) is inevitable with transplantation surgery, occur-
ring during organ resection, harvest, and graft implant, and
can also occur with trauma and hemorrhagic shock [1]. IRI
is a primary cause of early graft failure after transplant and
can lead to a higher incidence of early acute and also long
term chronic rejection. This ongoing injury to transplanted
organs contributes to significant graft loss after the first year
post transplant, creating the need for repeat transplantation
and is one cause for the acute shortages of donor organs
available for transplantation [1].
As ischemia-reperfusion injury is seen often during
transplantation, attention has been centered on identifying
methods to reduce IRI during and post-transplant (e.g.,
liver, lung, heart, and kidney transplants). For liver grafts,
the post-transplantation standard of care commonly
involves targeting IL-2 with either monoclonal antibodies
(e.g., Basiliximab) [2], mycophenolic acid [3]orFK506[4]
(frequently as a cocktail) in an effort to prevent T cell
proliferation and activity. Other approaches include inhib-
ition of the mammalian target of rapamycin (mTOR) with
drugs such as rapamycin and Everolimus [5], or general-
ized immune suppression with cyclosporine A or steroids
[6]. While these treatments have significantly improved
outcomes following liver transplantation in the past three
decades, many adverse effects persist. Most notably, trans-
plant recipients are at increased risk for malignancy [7],
viremia [8,9], bone loss [10], new-onset diabetes [11]and
cardiovascular disease [12], many of which are associated
with post-transplant immune suppression. Experimental
approaches to reduce post-ischemic injury while also
avoiding these adverse effects have included activation or
inactivation of specific signaling pathways by genetic or
small molecular perturbation [13–15], and pre- or post-
treatment with a variety of small molecules [16–18].
Despite experimental advances, movement towards the
clinic has been slow and there is a substantial need for
steroid-sparing, immune modulatory treatments designed
to prevent tissue loss after IRI.
Viruses have evolved advanced and highly potent
immune-modulating strategies over millions of years of
co-evolution with mammalian hosts. A key example of
one such virus is Myxomavirus, a leporipoxvirus and the
causative agent of a lethal infection, myxomatosis, in the
European rabbit (Oryctolagus cuniculus). Interestingly,
due to incompletely understood mechanisms, Myxoma-
virus is host-restricted to O. cuniculus and is not patho-
genic in other rabbit species and in humans [19].
Myxomavirus has evolved to a highly effective pathogen
in rabbits through development of potent immune modu-
lating proteins deployed to subvert, suppress and over-
whelm the host immune response. We have previously
demonstrated therapeutic benefit through delivery of
these immune modulators as either recombinant, purified
proteins or a coding sequence DNA in Adeno-associated
viral vectors (AAV) in animal model studies of disease.
For example, the Myxomavirus protein M-T7 is a
chemokine-GAG interaction inhibiting protein that re-
duces renal transplant rejection in both rats [20] and mice
[21], and decreases vascular balloon injury in rabbits and
rats [22]. In other work, we have demonstrated that treat-
ment with Serp-1, a member of the serpin superfamily of
proteins, as well as peptides derived from the Serp-1 react-
ive center loop (RCL), reduce severity and prolong sur-
vival in a lethal, herpesvirus-induced model of large vessel
vasculitis [23–25]. These and other examples demonstrate
that immune modulatory proteins employed by Myxoma-
virus for anti-immunological evasion are attractive pro-
teins for repurposing as new therapeutic approaches.
Serp-2 is a second Myxomavirus-derived serpin that is
a critical virulence factor for Myxomavirus. Viruses defi-
cient in Serp-2 cause robustly attenuated infections with
substantial increases in virus-limiting inflammation [26].
Early molecular work on Serp-2 has demonstrated
cross-class inhibitor activity for caspase-1 in the inflam-
masome signaling pathway, as well as caspases 8 and 10
and granzyme B in the apoptosis pathway [27–29]. Thus,
by inhibiting both inflammasome and apoptotic signal-
ing, Serp-2 enables Myxomavirus to suppress inflamma-
tion and avoid immune clearance.
In prior work, we tested Serp-2 treatment as an im-
mune modulatory, anti-inflammatory protein therapeutic
to reduce disease pathology in mouse models. A single
Yaron et al. Journal of Inflammation (2019) 16:12 Page 2 of 9
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
administration of Serp-2 treatment significantly reduced
aortic aneurysm formation and plaque growth in an aor-
tic angioplasty model in Apolipoprotein E-deficient
(ApoE
−/−
) mice over a period of 4 weeks [30]. In other
work, Serp-2 potently reduced plaque growth and in-
flammation in two separate models: a rat model of iliofe-
moral balloon angioplasty injury, as well as aortic
allograft transplant of plasminogen activator inhibitor 1-
deficient (PAI-1
−/−
) or ApoE
−/−
aortas into Balb/C re-
cipient mice [31]. Serp-2 lost activity in granzyme B/
ApoE double knock-out aortic allograft transplants.
Interestingly, in a carotid cuff injury model in ApoE
−/−
mice, Serp-2 displayed systemic effects against plaque
growth at the aortic root, a site distal to the acute cuff
injury [31]. Thus, Serp-2 has been demonstrated as an
effective and potent systemic, cross-class immune
modulator against tissue injury in a variety of inflamma-
tory in vivo models.
This short report extends prior studies with Serp-2 as
a virus-derived, therapeutic immune modulator to an
analysis of the potential for treatment with Serp-2 in a
mouse model for LIRI. Progression of LIRI has been at-
tributed to a variety of cellular mechanisms. Among the
proposed mechanisms, perturbation of the apoptotic and
inflammasome signaling cascades has demonstrated effi-
cacy in in vivo models [14,32–38]. On this basis, we hy-
pothesized that the apoptosis and inflammasome
inhibitory functions of Serp-2 would reduce pathology in
liver ischemia-reperfusion injury. Here, we investigated
LIRI as a controlled, outcomes-focused (i.e., survival)
model for testing further applicability of Serp-2 as a
therapeutic protein.
Methods
Mouse liver ischemia reperfusion injury (LIRI)
All animal protocols were approved by the University of
Florida Institutional Animal Care and Use Committee
(IACUC) and conform to national guidelines. All animals
received care in compliance with the Principles of Labora-
tory Animal Care and National standards. A total of 35
mice had liver ischemia reperfusion injury (LIRI) with 70%
surgical occlusion of the hepatic blood supply; five mice
had a sham operation. From the occlusion groups, 22 mice
had follow-up for 10 days (Saline, N= 10; Serp-2, N=8;
M-T7, N = 8) and 12 mice had follow-up at 24 h (Sham, N
= 5; LIRI Saline, N= 6; LIRI Serp-2, N= 3; LIRI M-T7, N =
3). A detailed description of mouse numbers and
treatments used in this study is given in Table 1. Serp-2 or
M-T7 (100 ng/g) in 100 μL saline was administered by in-
traperitoneal bolus through a 30-gauge needle, given 30
min prior to LIRI and then on alternate days for a total of
5 doses. Control mice received 100 μL saline in the same
regimen. All mice surviving to 10days were euthanized.
Warm, segmental ischemia to the left and middle hep-
atic lobes was performed as previously described [39].
Briefly, mice were anesthetized with a ketamine/xylazine
mixture. Buprenorphine was given subcutaneously (SC)
immediately prior to surgery and postoperatively as an
analgesic. After shaving and washing the abdominal area
with a three stage betadine soap/alcohol/betadine topical
wash, an incision was made using sterile technique from
the xiphoid process to the symphysis pubis and the por-
tal vein was exposed. An atraumatic clip was used to
interrupt the artery/portal venous blood supply to the
left and middle liver lobes (i.e., only the left and middle
hepatic artery and portal vein are occluded by the clip to
achieve 70% occlusion, while the right branch of portal
vein and hepatic artery are patent providing normal
blood flow). No intestinal ischemia was seen with this
model because the right branch of hepatic blood flow re-
mains open. Wet gauze was used to cover the incision
during IR injury. Blanching of the left and middle lobes
was observed as confirmation of ischemia. After 90 min
of ischemia, the clamp was removed, to allow reperfu-
sion, as confirmed by return of blood flow and return of
color. Saline (200–300 μL) was injected subcutaneously
as a resuscitation bolus at a site remote from the surgical
incision on the dorsum of the mouse. The inner muscle
and connective tissue were then closed with absorbent
suture (4–0 Coated VICRYL Polyglactin 910 Absorbable
Suture) and dermal layers closed with sterile nylon su-
ture. Sutures were removed at 7–10 days post-surgery.
Protein expression and purification
Serp-2 was His-tagged (His10) at the amino-terminus,
expressed from a vaccinia/T7 vector in HeLa cells and
purified as previously described [31]. M-T7 was expressed
from stabilized CHO cells as previously described [22,40].
Expressed proteins were immobilized for purification by
metal affinity using His-Bind resin (Novagen/Merck Milli-
pore, Burlington, MA, USA). Eluted proteins were found
to be > 90% pure by 12% SDS-PAGE following by silver
staining and immunoblotting.
Table 1 Numbers of mice
Treatment Follow-up # C57BL6/J Mice
Sham N/A 24 h 5
70% Ischemia-Reperfusion Saline 24 h 6
Saline 10 days 7
Serp-2 24 h 3
Serp-2 10 days 8
a
M-T7 24 h 3
M-T7 10 days 8
a
One of the original eight mice in this group was censored from analysis due
to post-surgical complications
Yaron et al. Journal of Inflammation (2019) 16:12 Page 3 of 9
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Serum alanine aminotransferase (ALT) measurement
Mice were euthanized at 24 h post-procedure and blood
was collected by cardiac puncture and allowed to clot
prior to centrifugation and storage of supernatant sera at
−80 °C until measurement. ALT was measured using a
kinetic, colorimetric diagnostic assay (#A7526, Pointe
Scientific, Canton, MI, USA). Briefly, 10 μL of serum
was measured in a total reaction volume of 100 μLby
the ALT-catalyzed transfer of the amino group from L-
alanine to α-ketoglutarate to form pyruvate and L-
glutamate and subsequent reduction of pyruvate and
oxidation of NADH to NAD by lactate dehydrogenase.
The biochemical reaction was monitored by reduction of
absorbance at 340 nm on a Molecular Devices M5e
multi-mode plate reader at 37 °C every minute for 5 min
in UV-transparent 96-well plates. The ΔOD340*min
−1
was converted to IU/L using a standard conversion
equation according to the manufacturer.
Histologic and morphometric analysis
At follow up, 24 h or 10 days, mice were euthanized, and
the liver harvested. For in-depth histological analysis we
used tissues from mice euthanized at 24 h (Sham, N=5;
LIRI Saline, N=6; LIRI Serp-2, N= 3; LIRI M-T7, N = 3).
Tissues were cut into 0.5 cm
3
sections for histological ana-
lysis. Liver sections were fixed in neutral buffered forma-
lin, paraffin embedded, cut into 4 μm cross sections, and
stained with hematoxylin and eosin or Periodic Acid-
Schiff. IRI infarct scar area and thickness at 24h follow up
as well as invading mononuclear cell counts were mea-
sured by morphometric analysis using an Olympus DP71
camera attached to an BX51 microscope (Olympus Amer-
ica Inc., Center Valley, PA, USA) and quantified using
Image Pro 6.0 (MediaCybernetics Inc., Bethesda, MD,
USA). Pathophysiologic histologic changes for LIRI were
evaluated by pathologists blinded to the treatment given
to each mouse based on the Suzuki scoring criteria [17].
Immunohistochemistry
FFPE sections were rehydrated through graded alcohol
and epitopes were retrieved by overnight incubation in
sodium citrate buffer at 60 °C. Sections were quenched
with 3% hydrogen peroxide in PBS for 15 min at room
temperature then blocked for 1 h with 5% BSA in TBST
at room temperature. Sections were probed overnight
with rabbit polyclonal to F4/80 (1:200 ab75476; Abcam,
Cambridge, MA, USA) followed by secondary goat-anti-
rabbit HRP-conjugated antibody (ab97051, Abcam) at a
dilution of 1:500. Immunoreactivity was revealed using
ImmPACT DAB (Vector Labs, Burlingame, CA, USA)
and sections were counterstained with hematoxylin,
dehydrated and mounted with Cytoseal XYL (Thermo
Scientific, Waltham, MA, USA). Positively stained cells
were counted in three high-power field areas (100× oil
immersion) in each liver cross section.
Immunoblotting
Frozen liver tissues were homogenized in RIPA lysis buf-
fer (Boston BioProducts) containing a 1× protease in-
hibitor cocktail (Bimake) using a blade homogenizer.
Homogenized samples were rotated at 4 °C for 2 h, pel-
leted at 15,000 gfor 20 min at 4 °C and supernatant
transferred to new tubes. Protein isolates were quantified
by BCA assay (Thermo Scientific), normalized with
RIPA buffer and boiled with 1× final concentration redu-
cing Laemmli buffer (Alfa Aesar) at 95 °C for 15 min.
Proteins (35 μg/sample) were resolved on a 15% SDS-
PAGE, transferred to a 0.2 μm pore PVDF membrane,
blocked with 5% non-fat dry milk in 0.1% TBS-Tween
20 and probed with primary antibodies against actin (1:
600, Rabbit polyclonal, Sigma Aldrich #A2066), cleaved
caspase-3 (1:1,000, Rabbit monoclonal, Cell Signaling #
9664S), caspase-8 (1:1,000, Mouse monoclonal, Protein-
tech #66093-1-IG) or caspase-1 (1:1,000, Mouse mono-
clonal, Adipogen #AG-20B-0042-C100) overnight in
blocking buffer at 4 °C with rocking. Secondary HRP-
conjugated antibodies against mouse (Jackson Immu-
noResearch #115-035-062) or rabbit (Jackson ImmunoR-
esearch #111-035-144) were incubated at room
temperature for 2 h in blocking buffer with shaking. Pro-
teins were revealed with Amersham ECL Start (actin;
GE #RPN3243) or ECL Prime (caspases 1, 3 and 8; GE
#RPN2236) on a GE LAS4000 imager on the high reso-
lution setting in 10 s increment developments until de-
sired image quality was achieved. Densitometry analysis
of cleaved caspase bands normalized to actin was per-
formed in Image Studio Lite v5.2.5 (Li-Cor Biosciences)
using the Top/Bottom averaging background correction
method with a border width of 3.
Statistical analysis
Statistical analysis was performed using GraphPad Prism
version 8 (GraphPad, La Jolla, CA, USA). Mean IR injury
area and cell count from three sections per infarcted
liver were analyzed by analysis of variance (ANOVA)
with Fishers PLSD (Protected Least Significant Differ-
ence) and unpaired, two-tailed Student’s T-test second-
ary analysis (p< 0.05 considered significant). Cumulative
survival was performed using the Kaplan-Meier survival
analysis with the Mantel-Cox statistical post-hoc test.
Results
Serp-2 treatment reduces acute injury and improves
survival in the mouse LIRI model
The effects of Serp-2 or M-T7 treatment on LIRI were
assessed after warm 70% occlusion of hepatic blood flow
for 90 min (Fig. 1A). Serp-2 treatment at a dose of 100 ng/g
Yaron et al. Journal of Inflammation (2019) 16:12 Page 4 of 9
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
delivered i.p. immediately prior to induction of LIRI signifi-
cantly reduced serum levels of alanine aminotransferase
(ALT), a clinical diagnostic marker for liver injury, at 24 h
(p= 0.0343). In comparison, M-T7, a virus-derived chemo-
kine inhibitor that has unrelated immune inhibitory func-
tions, when given at the same dose did not reduce ALT
levels when compared to saline (Fig. 1B). Serp-2 signifi-
cantly reduced mortality with 6 out of 7 C57BL6/J wildtype
(WT) mice surviving to 10 days with only one early loss at
56 h (p= 0.0135) when compared to saline treated WT
mice, in which 6 out of 7 mice died by 10 days (Fig. 1C).
M-T7 showed partial effectiveness with 4 of 7 mice survival
to 10 days, but this increased survival was not significant
(Fig. 1C). Thus, Serp-2 treatment alone was sufficient to
prolong short-term survival in mice post-LIRI.
Serp-2 treatment reduces post-ischemic liver injury
We next investigated the effect of Serp-2 or M-T7 treat-
ment on maintenance of liver viability after ischemia-
reperfusion. When compared to sham-operated mice,
histopathology clearly demonstrates a significant in-
crease in hepatocyte necrosis in saline-treated mice. This
tissue necrosis was ameliorated by treatment with Serp-
2, but not by M-T7 (Fig. 2A). Regions of the liver af-
fected by IRI that developed evidence for infarction and
scarring had significantly reduced infarct areas after
treatment with Serp-2, but not with M-T7 when com-
pared to saline at 24 h follow up (Fig. 2B; p= 0.0016). At
this point in our study, we determined that M-T7 was
not effective in this model and thus focused on the
mechanism and therapeutic benefit of Serp-2.
Independent histopathological analysis by pathologists
blinded to treatments, indicated that numerous indicators
of liver viability were improved with Serp-2 treatment
(Table 2). Compared to saline controls, Serp-2 treatment
significantly reduced liver necrosis (p= 0.0057) with a
strong trend towards significance in reducing hepatocyte
vacuolization (p= 0.0631) and a modest reduction in con-
gestion (p= 0.5128). Aggregate overall pathology score in-
dicated significant improvement with Serp-2 treatment (p
= 0.0046). Protection against worsened injury by Serp-2
treatment was not due to prevention of caspase-1, −3or
−8 cleavage (Additional file 1: Figure S1).
Serp-2 reduces early inflammatory infiltration to infarcted
post-ischemic liver tissue
We initially observed a reduction in the number of non-
specific inflammatory cells in Serp-2 treated livers by
H&E staining (small, dense nuclei). Macrophage-driven
inflammation has been previously reported to drive liver
ischemia-reperfusion injury in an inflammasome-
dependent manner [36]. Monocyte/macrophage infiltra-
tion into post-transplant livers is also associated with
worsened outcomes [41]. We thus investigated whether
Serp-2 suppressed invading macrophage counts after
LIRI. Immunohistochemical staining for the F4/80 pan-
macrophage antigen revealed a marked reduction in the
number of macrophages detected in the infarct scar zone
of post-ischemic livers at 24 h (Fig. 3;p= 0.0197).
Discussion
Despite advances in surgical procedure techniques and
post-transplant care and immunosuppression, IRI remains
a primary cause for early graft loss after liver transplant-
ation [1]. While questions remain as to the exact mechan-
ism of graft loss caused by LIRI, many groups have shown a
crucial role for apoptotic [42,43] and also inflammasome
pathway activation and signaling [14,34,36,38]. Study of
these pathways has led to substantially improved treat-
ments in other sterile diseases, and thus attention has now
been focused on developing similar treatments in LIRI.
Here, we investigate the potential for Serp-2, a
Myxomavirus-derived serine proteinase inhibitor (serpin)
with known cross-class inhibition of caspase-1 in the
Fig. 1 Serp-2 treatment improves survival following liver ischemia-reperfusion injury. (a) Experimental outline. Mice were treated with Serp-2, M-
T7 or saline (treatment; TX) 30 min prior to induction of 70% ischemia-reperfusion maintained for 90 min and were treated with Serp-2, M-T7 or
saline on alternating days for 10 days. (b) ALT levels in the serum of sham-operated mice or mice treated with saline, Serp-2 or M-T7 mice with
ischemia-reperfusion injury at 24 h post-procedure. Statistics calculated by one-way ANOVA with Fisher’s PLSD post-hoc analysis (N=2–6 mice
per group). (c) Mice treated with Serp-2 (magenta triangles) had significantly improved survival outcomes compared with mice treated with
saline, while mice treated with M-T7 (teal squares) did not show improved survival. Kaplan Meier curve statistics calculated by Log-rank
(Mantel-Cox) test (N=7–8 mice per group)
Yaron et al. Journal of Inflammation (2019) 16:12 Page 5 of 9
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
inflammasome signaling cascade and caspases 8 and 10
and granzyme B in the apoptosis signaling cascade, to
ameliorate LIRI severity in mice. We have demonstrated
here that while saline-treated mice die early after LIRI, re-
peated injections of Serp-2 (every alternate day) at a dose
of 100 ng/g, in the absence of other immune suppression
treatments, significantly prolonged survival for up to 10
days. Ten days was the endpoint of our study (Fig. 1C).
Protection against ischemia-reperfusion injury was likely
initiated early and not due to accelerated healing as ALT,
an indicator of acute liver injury, was reduced by Serp-2 at
24 h (Fig. 1B). The improved survival and reduced damage
were specific to the function of Serp-2. Similar same-dose
treatments with M-T7, an unrelated Myxomavirus-
derived immune modulating protein, did not provide any
survival advantage, reduction in acute injury markers nor
preservation of tissue viability (Figs. 1and 2). We also
note that not only do mice treated with Serp-2 survive,
but their livers display significant reductions in necrosis
and overall pathology when compared to saline-treated
mice (Fig. 2and Table 2). In investigating a potential
physiologic mechanism for Serp-2-dependent survival ad-
vantage in this model, we note that in addition to reduced
infarct scarring of the liver, areas of infarct had smaller in-
flammatory cell infiltrates, identified as macrophages by
immunohistochemistry (Fig. 3). While we cannot
specifically identify whether the macrophage infiltrates in
the infarcted region are so-called “cavity”macrophages
[44] or tissue-resident Kupffer cells, we note that the sur-
vival outcomes from Serp-2 treatment combined with the
reduction of invasive monocyte-lineage inflammatory cells
agrees with prior clinical studies [41].
Ischemia-reperfusion injury creates a complex physio-
logical state in the liver, involving hypoxia, reactive oxy-
gen and nitrogen species formation, multiple forms of
cell death and subsequent damage associate molecular
pattern (DAMP) release, all of which initiate a feed-
forward cascade of damage [45]. Indeed, the mechanism
of IRI in the liver remains a topic of active debate, par-
ticular with respect to the role of apoptosis, necrosis and
other forms of cell death in propagating post-ischemic
tissue damage [37,42,46,47]. Accordingly, caspase
cleavage has been reported even in the presence of a
number of other small molecule and biologic treatments
shown to preserve liver viability during warm IR proce-
dures [48–51]. Here, we found profound protection
against ischemia-reperfusion injury by treatment with
Serp-2, despite still observing caspase-1, −3 and −8 acti-
vation at 24 h follow-up (Additional file 1: Figure S1).
Our data therefore agree with the principle that activa-
tion of apoptotic and inflammatory caspases is not by it-
self a direct nor the sole indicator of tissue viability or
injury following ischemia-reperfusion, and that histo-
pathology or functional readouts (e.g., circulating
markers of injury such as ALT) are preferred for asses-
sing the effect of protection in this model [37]. It should
also be noted that Serp-2 may alter circulating levels of
proteases rather than tissue levels and that the tissue
isolates will represent a composite of multiple cell types
that may respond heterogeneously to the Serp-2 medi-
ated protective functions. Thus, the precise physiological
mechanism and the precise cell targets for Serp-2
Fig. 2 Serp-2 preserves tissue viability after ischemia-reperfusion injury. (a) Representative images of sham operated or LIRI-induced mice treated
with saline, Serp-2 or M-T7 with 2× or 10× objectives (20× and 100× magnification, respectively). 10× objective image regions are indicated by
black boxes in the 2× objective images. Scale bars represent 1000 μm (2× obj.) and 200 μm (10× obj.). Infarcted tissue is indicated with black
arrows. (b) Relative measure of infarct thickness in livers of LIRI-induced mice treated with saline, Serp-2 or M-T7. Statistics calculated by one-way
ANOVA with Fisher’s PLSD post-hoc test (N = 2–5 mice per group)
Table 2 Suzuki scores of mice treated with saline or Serp-2
Category Saline Serp-2 P-value
a
Congestion 2.000 ± 0.2532 1.700 ± 0.3958 0.5128
Vacuolization 2.692 ± 0.2083 2.000 ± 0.2981 0.0631
Necrosis 2.615 ± 0.2895 1.500 ± 0.1667 0.0057
Overall Pathology 2.437 ± 0.1157 1.734 ± 0.2035 0.0046
a
P-values were calculated by unpaired, two-tailed T-test. Significance
(p<0.05) is indicated by bolded text
Yaron et al. Journal of Inflammation (2019) 16:12 Page 6 of 9
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
protection against ischemia-reperfusion injury in the liver,
as described in this brief report, remains to be elucidated.
Further work will focus on potential extracellular effects
of Serp-2, such as inhibition of extracellular and circulat-
ing active caspases released from dying cells, a recently re-
ported mediator of inflammation amplification [52–54],
or on potential therapeutic benefit of Serp-2-derived me-
tabolites and peptides, such as we have described for
Serp-1, a related serpin from Myxomavirus [24,25].
Myxomavirus-derived proteins as therapeutic agents
have proven highly effective in a wide variety of animal
models [20,21,55,56]. Of importance, the evolution of
these proteins within poxvirus vectors has led these pro-
teins to exhibit very low immunogenicity. Indeed, Serp-1
was proven safe and effective in a Phase IIa clinical trial
in humans with acute unstable coronary syndrome [57].
Serp-1 dose-dependently reduced markers of heart dam-
age with a Major Adverse Cardiac Event (MACE) score
of zero and without induction of neutralizing antibody.
The safety and cross-species efficacy of Myxomavirus-
derived proteins, and in this study with Serp-2, high-
lights the potential for developing these agents for treat-
ment of inflammatory diseases. The substantial and
significant Serp-2 mediated therapeutic benefit post-LIRI
as demonstrated in this study indicates the potential for
Serp-2 treatment in liver transplantation. Further study
is warranted for testing Serp-2 and other Myxomaviral
proteins in preserving engrafted tissues.
Additional file
Additional file 1: Figure S1. Serp-2 mediated protection against LIRI at
24 hours does not prevent cleavage of caspases 1, 3 and 8. (a)Immunoblot
analysis of 2 mice each from sham surgery or from 90 minutes liver
ischemia-reperfusion injury at 24 hours follow-up probed for antibodies
against cleaved caspase-3 (p19), full length/cleaved caspase-8 (p45/p18) and
full length/cleaved caspase-1 (p45/p20) with actin as a loading control. (b)
Densitometry of cleaved bands for caspases-1, -3 and -8 normalized to actin.
Statistics performed by 2-Way ANOVA with Fisher’s LSD. (TIF 361 kb)
Abbreviations
AAV: Adeno-associated viral vectors; ALT: Alanine aminotransferase;
ApoE: Apolipoprotein E; H&E: hematoxylin and eosin; IP: intraperitoneal;
IRI: Ischemia-reperfusion injury; LIRI: Liver ischemia-reperfusion injury;
mTOR: mammalian target of rapamycin; PAI-1: plasminogen activator
inhibitor-1; SC: subcutaneous; serpin: serine proteinase inhibitor; WT: wildtype
Acknowledgements
The authors gratefully acknowledge Dr. Grant McFadden for excellent, critical
discussions.
Authors’contributions
Conceptualization: HC and ARL; Methodology: HC, JRY, ARL; Analysis:
HC, JRY, SA, LZ, BHM, ARL; Investigation: HC, JRY, SA, LZ, AMT;
Pathology: DW, JF, BJM, KH, WC, ARL; Resources: MYB, JD, DZ, KR;
Manuscript Preparation: JRY, BHM, LZ, ARL; Review: JRY, LZ, AMT, BHM,
ARL; Visualization: JRY, BHM, AMT, ARL; Supervision: ARL; Project
Administration: ARL; Funding Acquisition: ARL. All authors read and
approved the final manuscript.
Funding
This study was financially supported by grants from the NIH (1 R01
AI100987-01A1), American Heart Association (17GRNT33460327), University of
Florida Gatorade Fund (00115070) and start-up funds from the Biodesign In-
stitute at Arizona State University all to ARL.
Availability of data and materials
All data generated or analyzed during this study are included in this
published article.
Ethics approval and consent to participate
All animal protocols were approved by University of Florida Institutional
Animal Care and Use Committee (IACUC) and conform to national
guidelines. All animals received care in compliance with the Principles of
Laboratory Animal Care and National standards.
Fig. 3 Serp-2 treatment suppressed macrophage infiltration into post-ischemic infarct tissue. (a) Representative fields at 20× and 40×
magnification of a sham-operated liver and from post-ischemic infarcted tissue in livers from saline or Serp-2 treated mice stained with an
antibody against F4/80 and counterstained with hematoxylin. Dashed red region indicates infarcted tissue as determined by necrotic
hepatocytes. Dashed yellow region indicates inflammatory cell infiltrates. Black arrows indicate F4/80-positive cells. Red arrows indicate necrotic
hepatocytes. Scale bars are 100 μm (20×) and 50 μm (40×). (b) Percentages of F4/80-positive infiltrating cells per high-power field in the infarcted
tissue of livers from saline or Serp-2 treated mice. Bars represent mean and standard error. Statistics calculated by Student’s T-test
Yaron et al. Journal of Inflammation (2019) 16:12 Page 7 of 9
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Consent for publication
Not applicable.
Competing interests
ARL holds patents on the proteins Serp-2 and M-T7. All other authors declare
that they have no competing interests.
Author details
1
Center for Personalized Diagnostics and Center for Immunotherapy, Vaccines and
Virotherapy, Biodesign Institute, Arizona State University, Tempe, AZ, USA.
2
The
Department of Tumor Surgery, Second Hospital of Lanzhou University and The Key
Laboratory of the Digestive System Tumors of Gansu Province, Lanzhou, China.
3
Department of Pathology, University of Florida, Gainesville, FL, USA.
4
Divisions of
Cardiovascular Medicine and Rheumatology, Department of Medicine, University of
Florida, Gainesville, FL, USA.
5
Department of Molecular Genetics and Microbiology,
University of Florida, Gainesville, FL, USA.
Received: 19 December 2018 Accepted: 17 May 2019
References
1. Ricca L, Lemoine A, Cauchy F, Hamelin J, Sebagh M, Esposti DD, et al.
Ischemic postconditioning of the liver graft in adult liver transplantation.
Transplantation. 2015;99:1633–43.
2. Ramirez CB, Doria C, di Francesco F, DiFrancesco F, Iaria M, Kang Y, et al.
Basiliximab induction in adult liver transplant recipients with 93% rejection-
free patient and graft survival at 24 months. Transplant Proc. 2006;38:3633–
5. https://doi.org/10.1016/j.transproceed.2006.10.110.
3. Wiesner R, Rabkin J, Klintmalm G, McDiarmid S, Langnas A, Punch J, et
al. A randomized double-blind comparative study of mycophenolate
mofetil and azathioprine in combination with cyclosporine and
corticosteroids in primary liver transplant recipients. Liver Transplant.
2001;7:442–50.
4. Wiesner RH. A long-term comparison of tacrolimus (FK506) versus
cyclosporine in liver transplantation: a report of the United States FK506
study group. Transplantation. 1998;66:493–9http://www.ncbi.nlm.nih.gov/
pubmed/9734494.
5. Klintmalm GB, Nashan B. The role of mTOR inhibitors in liver transplantation:
reviewing the evidence. J Transp Secur. 2014;2014:1–45. https://doi.org/10.
1155/2014/845438.
6. Kotsch K, Ulrich F, Reutzel-Selke A, Pascher A, Faber W, Warnick P, et al.
Methylprednisolone therapy in deceased donors reduces inflammation in
the donor liver and improves outcome after liver transplantation a
prospective randomized controlled trial. Ann Surg. 2008;248:1042–9.
7. Fung JJ, Jain A, Kwak EJ, Kusne S, Dvorchik I, Eghtesad B. De novo
malignancies after liver transplantation: a major cause of late death. Liver
Transplant. 2001;7(11 SUPPL. 1):109–18.
8. Burak KW, Kremers WK, Batts KP, Wiesner RH, Rosen CB, Razonable RR, et al.
Impact of cytomegalovirus infection, year of transplantation, and donor age
on outcomes after liver transplantation for hepatitis C. Liver Transpl. 2002;8:
362–9. https://doi.org/10.1053/jlts.2002.32282.
9. Berenguer M, Ferrell L, Watson J, Prieto M, Kim M, Rayón M, et al. HCV-
related fibrosis progression following liver transplantation: increase in recent
years. J Hepatol. 2000;32:673–84.
10. Meys E, Fontanges E, Fourcade N, Thomasson A, Pouyet M, Delmas PD.
Bone loss after orthotopic liver transplantation. Am J Med. 1994;97:445–50.
11. Marchetti P. New-onset diabetes after liver transplantation: from
pathogenesis to management. Liver Transplant. 2005;11:612–20.
12. Fussner LA, Heimbach JK, Fan C, Dierkhising R, Coss E, Leise MD, et al.
Cardiovascular disease after liver transplantation: when, what, and who
is at risk. Liver Transpl. 2015;21:889–96. https://doi.org/10.1002/lt.24137.
13. Suetsugu H, Iimuro Y, Uehara T, Nishio T, Harada N, Yoshida M, et al.
Nuclear factor κB inactivation in the rat liver ameliorates short term total
warm ischaemia/reperfusion injury. Gut. 2005;54:835–42.
14. Zhu P, Duan L, Chen J, Xiong A, Xu Q, Zhang H, et al. Gene silencing of
NALP3 protects against liver ischemia–reperfusion injury in mice. Hum Gene
Ther. 2011;22:853–64. https://doi.org/10.1089/hum.2010.145.
15. Zhang W, Zhang J, Mulholland M, Zhang W. mTOR activation protects liver
from ischemia/reperfusion-induced injury through NF-κB pathway. FASEB J.
2017;31:3018–26.
16. Duenschede F, Erbes K, Kircher A, Westermann S, Schad A, Riegler N, et al.
Protection from hepatic ischemia/reperfusion injury and improvement of
liver regeneration by α-lipoic acid. Shock. 2007;27:644–51.
17. Suzuki S, Toledo-Pereyra LH, Rodriguez FJ, Cejalvo D. Neutrophil infiltration
as an important factor in liver ischemia and reperfusion injury. Modulating
effects of FK506 and cyclosporine. Transplantation. 1993;55:1265–72. https://
doi.org/10.1097/00007890-199306000-00011.
18. Takasu C, Vaziri ND, Li S, Robles L, Vo K, Takasu M, et al. Treatment with
dimethyl fumarate ameliorates liver ischemia/reperfusion injury. World J
Gastroenterol. 2017;23:4508. https://doi.org/10.3748/wjg.v23.i25.4508.
19. Best SM, Kerr PJ. Coevolution of host and virus: the pathogenesis of virulent
and attenuated strains of myxoma virus in resistant and susceptible European
rabbits. Virology. 2000;267:36–48. https://doi.org/10.1006/viro.1999.0104.
20. Bedard EL, Jiang J, Arp J, Qian H, Wang H, Guan H, et al. Prevention of chronic
renal allograft rejection by SERP-1 protein. Transplantation. 2006;81:908–14.
21. Chen H, Ambadapadi S, Wakefield D, Bartee M, Yaron JR, Zhang L, et
al. Selective deletion of Heparan sulfotransferase enzyme, Ndst1, in
donor endothelial and myeloid prec ursor cell s significantly decreases
acute allograft rejection. Sci Rep. 2018;8:1–16. https://doi.org/10.1038/
s41598-018-31779-7.
22. Liu L, Lalani A, Dai E, Seet B, Macauley C, Singh R, et al. The viral anti-
inflammatory chemokine-binding protein M-T7 reduces intimal hyperplasia after
vascular injury. J Clin Invest. 2000;105:1613–21. https://doi.org/10.1172/JCI8934.
23. Chen H, Zheng D, Abbott J, Liu L, Bartee MY, Long M, et al. Myxomavirus-derived
serpin prolongs survival and reduces inflammation and hemorrhage in an unrelated
lethal mouse viral infection. Antimicrob Agents Chemother. 2013;57:4114–27.
24. Ambadapadi S, Munuswamy-Ramanujam G, Zheng D, Sullivan C, Dai E,
Morshed S, et al. Reactive center loop (RCL) peptides derived from serpins
display independent coagulation and immune modulating activities. J Biol
Chem. 2016;291:2874–87.
25. Mahon BP, Ambadapadi S, Yaron JR, Lomelino CL, Pinard MA, Keinan S, et
al. Crystal structure of cleaved Serp-1, a Myxomavirus-derived immune
modulating serpin: structural Design of Serpin Reactive Center Loop
Peptides with improved therapeutic function. Biochemistry. 2018;57:1096–
107. https://doi.org/10.1021/acs.biochem.7b01171.
26. Messud-Petit F, Gelfi J, Delverdier M, Amardeilh MF, Py R, Sutter G, et al.
Serp2, an inhibitor of the interleukin-1beta-converting enzyme, is critical in
the pathobiology of myxoma virus. J Virol. 1998;72:7830–9. https://www.
ncbi.nlm.nih.gov/pubmed/9733819.
27. Petit F, Bertagnoli S, Gelfi J, Fassy F, Boucraut-Baralon C, Milon A.
Characterization of a myxoma virus-encoded serpin-like protein with activity
against interleukin-1 beta-converting enzyme. J Virol. 1996;70:5860–6http://
www.ncbi.nlm.nih.gov/pubmed/8709205.
28. Turner PC, Sancho MC, Thoennes SR, Caputo A, Bleackley RC, Moyer RW.
Myxoma virus Serp2 is a weak inhibitor of granzyme B and interleukin-
1beta-converting enzyme in vitro and unlike CrmA cannot block apoptosis
in cowpox virus-infected cells. J Virol. 1999;73:6394–404. https://www.ncbi.
nlm.nih.gov/pubmed/10400732.
29. MacNeill AL, Turner PC, Moyer RW. Mutation of the Myxoma virus SERP2 P1-
site to prevent proteinase inhibition causes apoptosis in cultured RK-13 cells
and attenuates disease in rabbits, but mutation to alter specificity causes
apoptosis without reducing virulence. Virology. 2006;356:12–22.
30. Davids JA, Dai E, Chen H, Bartee MY, Liu L, Fortunel A, et al. Viral anti-
inflammatory proteins target diverging immune pathways with converging
effects on arterial dilatation, plaque and apoptosis. Eur J Inflamm. 2014;12:
131–45. https://doi.org/10.1177/1721727X1401200113.
31. Viswanathan K, Bot I, Liu L, Dai E, Turner PC, Togonu-Bickersteth B, et al.
Viral cross-class serpin inhibits vascular inflammation and T lymphocyte
fratricide; a study in rodent models in vivo and human cell lines in vitro.
PLoS One. 2012;7.
32. Sadatomo A, Inoue Y, Ito H, Karasawa T, Kimura H, Watanabe S, et al.
Interaction of neutrophils with macrophages promotes IL-1βmaturation
and contributes to hepatic ischemia–reperfusion injury. J Immunol. 2017:
ji1700717. https://doi.org/10.4049/jimmunol.1700717.
33. Sun K, Liu ZS, Sun Q. Role of mitochondria in cell apoptosis during hepatic
ischema-reperfusion injury and protective effect of ischemic
postconditioning. World J Gastroenterol. 2004;10:1934–8.
34. Huang H, Chen H-W, Evankovich J, Yan W, Rosborough BR, Nace GW, et al.
Histones activate the NLRP3 Inflammasome in Kupffer cells during sterile
inflammatory liver injury. J Immunol. 2013;191:2665–79. https://doi.org/10.
4049/jimmunol.1202733.
Yaron et al. Journal of Inflammation (2019) 16:12 Page 8 of 9
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
35. Kato A, Gabay C, Okaya T, Lentsch AB. Specific role of interleukin-1 in
hepatic neutrophil recruitment after ischemia/reperfusion. Am J Pathol.
2002;161:1797–803. https://doi.org/10.1016/S0002-9440(10)64456-2.
36. Kamo N, Ke B, Ghaffari AA, da Shen X, Busuttil RW, Cheng G, et al. ASC/
caspase-1/IL-1βsignaling triggers inflammatory responses by promoting
HMGB1 induction in liver ischemia/reperfusion injury. Hepatology. 2013;58:
351–62.
37. Gujral JS, Bucci TJ, Farhood A, Jaeschke H. Mechanism of cell death during
warm hepatic ischemia-reperfusion in rats: apoptosis or necrosis?
Hepatology. 2001;33:397–405. https://doi.org/10.1053/jhep.2001.22002.
38. Kim HY, Kim SJ, Lee SM. Activation of NLRP3 and AIM2 inflammasomes in
Kupffer cells in hepatic ischemia/reperfusion. FEBS J. 2015;282:259–70.
39. Tsung A, Hoffman RA, Izuishi K, Critchlow ND, Nakao A, Chan MH, et al.
Hepatic ischemia/reperfusion injury involves functional TLR4 signaling in
nonparenchymal cells. J Immunol. 2005;175:7661–8. https://doi.org/10.4049/
jimmunol.175.11.7661.
40. Bartee MY, Chen H, Dai E, Liu LY, Davids JA, Lucas A. Defining the anti-
inflammatory activity of a potent myxomaviral chemokine modulating
protein, M-T7, through site directed mutagenesis. Cytokine. 2014;65:79–87.
41. Robertson F, Male V, Wright G, Fuller B, Davidson B. Recruitment of
inflammatory monocytes after liver transplantation and correlation with
clinical outcome. Lancet. 2017;389:S84. https://doi.org/10.1016/S0140-
6736(17)30480-4.
42. Jaeschke H, Lemasters JJ. Apoptosis versus oncotic necrosis in hepatic
ischemia/reperfusion injury. Gastroenterology. 2003;125:1246–57. https://doi.
org/10.1016/S0016-5085(03)01209-5.
43. Tsung A, Kaizu T, Nakao A, Shao L, Bucher B, Fink MP, et al. Ethyl pyruvate
ameliorates liver ischemia-reperfusion injury by decreasing hepatic necrosis
and apoptosis. Transplantation. 2005;79:196–204.
44. Wang J, Kubes P. A reservoir of mature cavity macrophages that can rapidly
invade visceral organs to affect tissue repair. Cell. 2016;165:668–78. https://
doi.org/10.1016/j.cell.2016.03.009.
45. Wu MY, Yiang GT, Liao WT, Tsai APY, Cheng YL, Cheng PW, et al. Current
mechanistic concepts in ischemia and reperfusion injury. Cell Physiol
Biochem. 2018;46:1650–67.
46. Schulze-Bergkamen H, Schuchmann M, Fleischer B, Galle PR. The role of
apoptosis versus oncotic necrosis in liver injury: facts or faith? J Hepatol.
2006;44:984–93. https://doi.org/10.1016/j.jhep.2006.02.004.
47. Clavien PA, Rüdiger HA, Selzner M. Mechanism of hepatocyte death after
ischemia: apoptosis versus necrosis. Hepatology. 2001;33:1555–7. https://doi.
org/10.1053/jhep.2001.0103306le02.
48. Kaizu T, Ikeda A, Nakao A, Takahashi Y, Tsung A, Kohmoto J, et al. Donor
graft adenoviral iNOS gene transfer ameliorates rat liver transplant
preservation injury and improves survival. Hepatology. 2006;43:464–73.
49. Zhu M, Lu B, Cao Q, Wu Z, Xu Z, Li W, et al. IL-11 attenuates liver ischemia/
reperfusion injury (IRI) through STAT3 signaling pathway in mice. PLoS One.
2015;10:1–15.
50. Song H, Du C, Wang X, Zhang J, Shen Z. MicroRNA-101 inhibits autophagy
to alleviate liver ischemia/reperfusion injury via regulating the mTOR
signaling pathway. Int J Mol Med. 2019;43:1331–42. https://doi.org/10.3892/
ijmm.2019.4077.
51. Xu Y, Yao J, Zou C, Zhang H, Zhang S, Liu J, et al. Asiatic acid protects
against hepatic ischemia/reperfusion injury by inactivation of Kupffer cells
via PPARγ/NLRP3 inflammasome signaling pathway. Oncotarget. 2017;8:
86339–55. https://doi.org/10.18632/oncotarget.21151.
52. Franklin BS, Bossaller L, De Nardo D, Ratter JM, Stutz A, Engels G, et al. The
adaptor ASC has extracellular and “prionoid”activities that propagate
inflammation. Nat Immunol. 2014;15:727–37.
53. Wang L, Fu H, Nanayakkara G, Li Y, Shao Y, Johnson C, et al. Novel
extracellular and nuclear caspase-1 and inflammasomes propagate
inflammation and regulate gene expression: a comprehensive database
mining study. J Hematol Oncol. 2016;9:1–18. https://doi.org/10.1186/
s13045-016-0351-5.
54. Hentze H, Schwoebel F, Lund S, Kehl M, Ertel W, Wendel A, et al. In vivo
and in vitro evidence for extracellular caspase activity released from
apoptotic cells. Biochem Biophys Res Commun. 2001;283:1111–7.
55. Dai E, Liu LY, Wang H, McIvor D, Sun YM, Macaulay C, et al. Inhibition of
chemokine-glycosaminoglycan interactions in donor tissue reduces mouse
allograft vasculopathy and transplant rejection. PLoS One. 2010;5.
56. Bédard ELR, Kim P, Jiang J, Parry N, Liu L, Wang H, et al. Chemokine-binding
viral protein M-T7 prevents chronic rejection in rat renal allografts.
Transplantation. 2003;76:249–52.
57. Tardif J-C, L’Allier PL, Grégoire J, Ibrahim R, McFadden G, Kostuk W, et al. A
randomized controlled, phase 2 trial of the viral serpin Serp-1 in patients
with acute coronary syndromes undergoing percutaneous coronary
intervention. Circ Cardiovasc Interv. 2010;3:543–8. https://doi.org/10.1161/
CIRCINTERVENTIONS.110.953885.
Publisher’sNote
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Yaron et al. Journal of Inflammation (2019) 16:12 Page 9 of 9
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
1.
2.
3.
4.
5.
6.
Terms and Conditions
Springer Nature journal content, brought to you courtesy of Springer Nature Customer Service Center GmbH (“Springer Nature”).
Springer Nature supports a reasonable amount of sharing of research papers by authors, subscribers and authorised users (“Users”), for small-
scale personal, non-commercial use provided that all copyright, trade and service marks and other proprietary notices are maintained. By
accessing, sharing, receiving or otherwise using the Springer Nature journal content you agree to these terms of use (“Terms”). For these
purposes, Springer Nature considers academic use (by researchers and students) to be non-commercial.
These Terms are supplementary and will apply in addition to any applicable website terms and conditions, a relevant site licence or a personal
subscription. These Terms will prevail over any conflict or ambiguity with regards to the relevant terms, a site licence or a personal subscription
(to the extent of the conflict or ambiguity only). For Creative Commons-licensed articles, the terms of the Creative Commons license used will
apply.
We collect and use personal data to provide access to the Springer Nature journal content. We may also use these personal data internally within
ResearchGate and Springer Nature and as agreed share it, in an anonymised way, for purposes of tracking, analysis and reporting. We will not
otherwise disclose your personal data outside the ResearchGate or the Springer Nature group of companies unless we have your permission as
detailed in the Privacy Policy.
While Users may use the Springer Nature journal content for small scale, personal non-commercial use, it is important to note that Users may
not:
use such content for the purpose of providing other users with access on a regular or large scale basis or as a means to circumvent access
control;
use such content where to do so would be considered a criminal or statutory offence in any jurisdiction, or gives rise to civil liability, or is
otherwise unlawful;
falsely or misleadingly imply or suggest endorsement, approval , sponsorship, or association unless explicitly agreed to by Springer Nature in
writing;
use bots or other automated methods to access the content or redirect messages
override any security feature or exclusionary protocol; or
share the content in order to create substitute for Springer Nature products or services or a systematic database of Springer Nature journal
content.
In line with the restriction against commercial use, Springer Nature does not permit the creation of a product or service that creates revenue,
royalties, rent or income from our content or its inclusion as part of a paid for service or for other commercial gain. Springer Nature journal
content cannot be used for inter-library loans and librarians may not upload Springer Nature journal content on a large scale into their, or any
other, institutional repository.
These terms of use are reviewed regularly and may be amended at any time. Springer Nature is not obligated to publish any information or
content on this website and may remove it or features or functionality at our sole discretion, at any time with or without notice. Springer Nature
may revoke this licence to you at any time and remove access to any copies of the Springer Nature journal content which have been saved.
To the fullest extent permitted by law, Springer Nature makes no warranties, representations or guarantees to Users, either express or implied
with respect to the Springer nature journal content and all parties disclaim and waive any implied warranties or warranties imposed by law,
including merchantability or fitness for any particular purpose.
Please note that these rights do not automatically extend to content, data or other material published by Springer Nature that may be licensed
from third parties.
If you would like to use or distribute our Springer Nature journal content to a wider audience or on a regular basis or in any other manner not
expressly permitted by these Terms, please contact Springer Nature at
onlineservice@springernature.com
Available via license: CC BY
Content may be subject to copyright.
