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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/6 J) 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). 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.
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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 [1315], and pre- or post-
treatment with a variety of small molecules [1618].
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 [2325]. 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 [2729]. 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 [14,3238]. 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 (200300 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 (40 Coated VICRYL Polyglactin 910 Absorbable
Suture) and dermal layers closed with sterile nylon su-
ture. Sutures were removed at 710 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
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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 Students 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
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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 Fishers PLSD post-hoc analysis (N=26 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=78 mice per group)
Yaron et al. Journal of Inflammation (2019) 16:12 Page 5 of 9
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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 cavitymacrophages
[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 [4851]. 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 Fishers PLSD post-hoc test (N = 25 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 [5254],
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 Fishers 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.
Authorscontributions
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 Students 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
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... In mouse models of aortic allograft transplants, Serp-2 significantly reduced inflammation and intimal hyperplasia, again with no detected side effects [50,51]. In a model of partial 70% warm ischemiareperfusion injury in the liver (LIRI), Serp-2 treatment given systemically also improved survival over 10 days, reduced necrotic damage of the liver and lowered acute markers of liver damage [61]. Surprisingly, caspase-1, caspase-3 and caspase-8 activation were not suppressed, suggesting an alternative mechanism of protection potentially by inhibition of circulating inflammatory proteases. ...
... Continuous infusion of vMIP-II decreased infiltrating hematogenous cells at the site of injury in a spinal cord contusion injury in rats, with associated reductions in neuronal loss and gliosis [150]. However, this study did not include an assessment of locomotor function, thus it remains to be seen whether chemokine antagonism with vMIP-II improves functional recovery, as seen with the poxvirus chemokine modulator M-T7 [61]. Gene transfer of vMIP-II by direct injection of plasmid DNA improved cardiac allograft survival when hearts were placed in the subcutaneous position of the ear pinnae of CBA/J recipients [151]. ...
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Viruses are widely used as a platform for the production of therapeutics. Vaccines containing live, dead and components of viruses, gene therapy vectors and oncolytic viruses are key examples of clinically-approved therapeutic uses for viruses. Despite this, the use of virus-derived proteins as natural sources for immune modulators remains in the early stages of development. Viruses have evolved complex, highly effective approaches for immune evasion. Originally developed for protection against host immune responses, viral immune-modulating proteins are extraordinarily potent, often functioning at picomolar concentrations. These complex viral intracellular parasites have "performed the R&D", developing highly effective immune evasive strategies over millions of years. These proteins provide a new and natural source for immune-modulating therapeutics, similar in many ways to penicillin being developed from mold or streptokinase from bacteria. Virus-derived serine proteinase inhibitors (serpins), chemokine modulating proteins, complement control, inflammasome inhibition, growth factors (e.g., viral vascular endothelial growth factor) and cytokine mimics (e.g., viral interleukin 10) and/or inhibitors (e.g., tumor necrosis factor) have now been identified that target central immunological response pathways. We review here current development of virus-derived immune-modulating biologics with efficacy demonstrated in pre-clinical or clinical studies, focusing on pox and herpesviruses-derived immune-modulating therapeutics.
... In other models, Serp-2 was found to suppress inflammation and improve outcomes in models of aortic transplant and carotid cuff compression injury [16][17][18]. Based on the overlap of the targeted protease pathways (inflammasome and apoptosis), Serp-2 was tested as a putative therapeutic to prevent liver injury and improve outcomes after 90 min of partial (70%) warm ischemia-reperfusion injury [19]. Using this model, we found that treatment with recombinant purified Serp-2 improves 10-day survival, ameliorates acute injury as measured by circulating liver markers, and preserves liver viability. ...
... We previously tested this model with purified recombinant Serp-2 protein [19]. To produce the recombinant protein, the coding sequence for Serp-2 was tagged with an N-terminal 10Â-histidine affinity tag and transformed into HeLa cells with a vaccinia/T7 vector [17,23]. ...
Chapter
Ischemia-reperfusion injury (IRI) drives early and long-term damage to organs as well as compounding damage from acute transplant rejection and surgical trauma. IRI initiates an aggressive and prolonged inflammation leading to tissue injury, organ failure, and death. However, there are few effective therapeutic interventions for IRI. The destructive inflammatory cell activity in IRI is part of an aberrant innate immune response that triggers multiple pathways. Hence, immune-modulating treatments to control pathways triggered by IRI hold great therapeutic potential. Viruses, especially large DNA viruses, have evolved highly effective immune-modulating proteins for the purpose of immune evasion and to protect the virus from the host immune defenses. A number of these immune-modulating proteins have proven therapeutically effective in preclinical models, many with function targeting pathways known to be involved in IRI. The use of virus-derived immune-modulating proteins thus represents a promising source for new treatments to target ischemia-reperfusion injury. Laboratory small animal models of IRI are well established and are able to reproduce many aspects of ischemia-reperfusion injury seen in humans. This chapter will discuss the methods used to perform the IRI procedure in mice, as well as clinically relevant diagnostic tests to evaluate liver injury and approaches for assessing histological damage while testing novel immune modulating protein treatments.
... Moreover, specific inhibitors of inflammasome need to be examined under conditions of different warm ischemia times and percentages. A recent study shows that treatment with Serp-2, a virus-derived inhibitor of apoptosis and inflammasome, regulates the levels of caspase-1, 8 and 10, improving the survival of mice submitted to 90 min of partial (70%) warm ischemia [128]. Interestingly, a study by Yang et al., using an experimental model of hepatic I/R based on 45 min of 70% warm ischemia, showed that Z-VD-fmk, a pan-caspase inhibitor (including caspase-1), had no effect on I/R injury or on the number of TUNEL-positive cells and staining pattern (nucleus and cytosol) [129]. ...
... Interestingly, a study by Yang et al., using an experimental model of hepatic I/R based on 45 min of 70% warm ischemia, showed that Z-VD-fmk, a pan-caspase inhibitor (including caspase-1), had no effect on I/R injury or on the number of TUNEL-positive cells and staining pattern (nucleus and cytosol) [129]. The authors noted the minor role of apoptosis, thus contesting a relevant role for inflammasome or pyroptosis in hepatic I/R, at least in conditions of 90 min of partial (70%) warm ischemia [128] or 30 min of total warm I/R injury [126]. Data suggest that future research should be focused on detailing the type of cell death (necrosis, apoptosis and/or pyroptosis) and the signaling mechanisms of cell death, to identify specific targets for attenuating hepatic I/R injury. ...
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Ischemia-reperfusion injury is an important cause of liver damage occurring during surgical procedures including hepatic resection and liver transplantation, and represents the main underlying cause of graft dysfunction and liver failure post-transplantation. To date, ischemia-reperfusion injury is an unsolved problem in clinical practice. In this context, inflammasome activation, recently described during ischemia-reperfusion injury, might be a potential therapeutic target to mitigate the clinical problems associated with liver transplantation and hepatic resections. The present review aims to summarize the current knowledge in inflammasome-mediated inflammation, describing the experimental models used to understand the molecular mechanisms of inflammasome in liver ischemia-reperfusion injury. In addition, a clear distinction between steatotic and non-steatotic livers and between warm and cold ischemia-reperfusion injury will be discussed. Finally, the most updated therapeutic strategies, as well as some of the scientific controversies in the field will be described. Such information may be useful to guide the design of better experimental models, as well as the effective therapeutic strategies in liver surgery and transplantation that can succeed in achieving its clinical application.
... In addition, the liver sections were analyzed according to the scoring system of Suzuki that assesses three main injury outcomes, congestion, vacuolization and necrosis (Suzuki et al., 1993). High levels of necrosis were evident in the livers of mice subjected to IR, consistent with previous findings (Yaron et al., 2019), and this was significantly reduced in the IR animals receiving AC pretreatment ( Figure 7B). In all analyses the readers were blinded to the treatment groups. ...
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... In a related study, Zhang et al. described that A1AT-dependent protection of islet viability after cytokine-and streptozotocininduced diabetes in mice was in part due to dramatic reduction of beta cell apoptosis (175) Ischemia-reperfusion injury is characterized by a transient loss of blood and oxygen to a tissue, followed by a period of reoxygenation which paradoxically accelerates damage caused during the hypoxic period (184). Ischemia-reperfusion injury can occur in any tissue, whether by pathogenic etiology or by complications of surgical procedure, and there is an unmet need for novel therapeutics to address the condition (185)(186)(187)(188). Moldthan et al. first demonstrated the therapeutic efficacy of A1AT therapy in a rat model of ischemic stroke which resulted in a drastic reduction of infarct volume and preservation of sensory motor system function (189). ...
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... Inflammasome adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD domain) deficiency, IL-1β blocking antibody injection [43], injection of Serp 2 (a virus-derived serine protease and pan-caspase inhibitor) [47], and caspase 1/caspase 4 substrate Gasdermin D deficiency [48] lead to protection against liver IRI [49]. Caspase 1 deficient mice are less susceptible to acetaminophen-induced liver injury [50]. ...
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... Myxoma virus (MYXV) is a leporipoxvirus with well-known strict species-specificity and hosttropism to the European rabbit (Oryctolagus cuniculus) [16]. We have demonstrated the safety and immunotherapeutic efficacy of several MYXV immune modulators in a wide array of preclinical models [17][18][19][20][21][22][23][24][25][26][27]. M-T7 is an MYXV-derived immune modulator with broad chemokine-binding activity and proven therapeutic potential in inflammation-related diseases. ...
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Myxoma virus (MYXV) has proven to be an effective candidate for oncolytic virotherapy in many preclinical cancer models. As a nonhuman pathogen, MYXV does not need to overcome any preexisting antiviral immunity, and its DNA cannot integrate into the host genome, making it an extremely safe vector. Moreover, the large dsDNA genome of MYXV allows the insertion of multiple transgenes and the design of engineered recombinant oncolytic viruses (OVs) with enhanced immunostimulatory or other desired properties. In this chapter, we describe detailed protocols for the generation and characterization of transgene-armed recombinant MYXV vectors.
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Ischemia-reperfusion injury is associated with serious clinical manifestations, including myocardial hibernation, acute heart failure, cerebral dysfunction, gastrointestinal dysfunction, systemic inflammatory response syndrome, and multiple organ dysfunction syndrome. Ischemia-reperfusion injury is a critical medical condition that poses an important therapeutic challenge for physicians. In this review article, we present recent advances focusing on the basic pathophysiology of ischemia-reperfusion injury, especially the involvement of reactive oxygen species and cell death pathways. The involvement of the NADPH oxidase system, nitric oxide synthase system, and xanthine oxidase system are also described. When the blood supply is re-established after prolonged ischemia, local inflammation and ROS production increase, leading to secondary injury. Cell damage induced by prolonged ischemia-reperfusion injury may lead to apoptosis, autophagy, necrosis, and necroptosis. We highlight the latest mechanistic insights into reperfusion-injury-induced cell death via these different processes. The interlinked signaling pathways of cell death could offer new targets for therapeutic approaches. Treatment approaches for ischemia-reperfusion injury are also reviewed. We believe that understanding the pathophysiology ischemia-reperfusion injury will enable the development of novel treatment interventions.
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The Myxomavirus-derived protein Serp-1 has potent anti-inflammatory activity in models of vasculitis, lupus, viral sepsis, and transplant. Serp-1 has also been tested successfully in a Phase IIa clinical trial in unstable angina, representing a 'first-in-class' therapeutic. Recently, peptides derived from the reactive center loop (RCL) have been developed as stand-alone therapeutics for reducing vasculitis and improving survival in MHV68-infected mice. However, both Serp-1 and the RCL peptides lose activity in MHV68-infected mice after antibiotic suppression of intestinal microbiota. Here, we utilize a structure-guided approach to design and test a series of next generation RCL peptides with improved therapeutic potential that is not reduced when combined with antibiotic treatments. The crystal structure of the cleaved Serp-1 was determined to 2.5 Å resolution, and reveals a classical serpin structure with potential for serpin-derived RCL peptides to bind and inhibit mammalian serpins, plasminogen activator inhibitor 1 (PAI-1), anti-thrombin III (ATIII) and alpha-1 antitrypsin (A1AT) and target proteases. Using in silico modeling of Serp-1 RCL peptide, S-7, we designed several modified RCL peptides that were predicted to have stronger interactions with human serpins due to a higher number of stabilizing hydrogen bonds. Two of these peptides (MPS7-8 and -9) displayed extended activity, improving survival where activity was previously lost in antibiotic treated MHV68-infected mice (P < 0.0001). Mass spectrometry and kinetic assays suggest peptide interaction with ATIII, A1AT, and target proteases in mouse and human plasma. In summary, we present the next step towards the development of a promising new class of anti-inflammatory serpin-based therapeutic.
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Accumulating evidence suggests that IL-1β plays a pivotal role in the pathophysiology of hepatic ischemia-reperfusion (I/R) injury; however, the mechanism by which I/R triggers IL-1β production in the liver remains unclear. Recent data have shown that neutrophils contribute to hepatic I/R injury independently of the inflammasomes regulating IL-1β maturation. Thus, we investigated the role of neutrophils in IL-1β maturation and tissue injury in a murine model of hepatic I/R. IL-1β was released from the I/R liver and its deficiency reduced reactive oxygen species generation, apoptosis, and inflammatory responses, such as inflammatory cell infiltration and cytokine expression, thereby resulting in reduced tissue injury. Depletion of either macrophages or neutrophils also attenuated IL-1β release and hepatic I/R injury. In vitro experiments revealed that neutrophil-derived proteinases process pro-IL-1β derived from macrophages into its mature form independently of caspase-1. Furthermore, pharmacological inhibition of serine proteases attenuated IL-1β release and hepatic I/R injury in vivo. Taken together, the interaction between neutrophils and macrophages promotes IL-1β maturation and causes IL-1β-driven inflammation in the I/R liver. Both neutrophils and macrophages are indispensable in this process. These findings suggest that neutrophil-macrophage interaction is a therapeutic target for hepatic I/R injury and may also provide new insights into the inflammasome-independent mechanism of IL-1β maturation in the liver.
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Hepatic ischemia/reperfusion (I/R) contributes to major complications in clinical practice affecting perioperative morbidity and mortality. Recent evidence suggests the key role of nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) inflammaosme activation on the pathogenesis of I/R injury. Asiatic acid (AA) is a pentacyclic triterpene derivative presented with versatile activities, including antioxidant, anti-inflammation and hepatoprotective effects. This study was designed to determine whether AA had potential hepatoprotective benefits against hepatic I/R injury, as well as to unveil the underlying mechanisms involved in the putative effects. Mice subjected to warm hepatic I/R, and Kupffer cells (KCs) or RAW264.7 cells challenged with lipopolysaccharide (LPS)/H2O2, were pretreated with AA. Administration of AA significantly attenuated hepatic histopathological damage, global inflammatory level, apoptotic signaling level, as well as NLRP3 inflammasome activation. These effects were correlated with increased expression of peroxisome proliferator-activated receptor gamma (PPARγ). Conversely, pharmacological inhibition of PPARγ by GW9662 abolished the protective effects of AA on hepatic I/R injury and in turn aggravated NLRP3 inflammasome activation. Activation of NLRP3 inflammasome was most significant in nonparenchymal cells (NPCs). Depletion of KCs by gadolinium chloride (GdCl3) further attenuated the detrimental effects of GW9662 on hepatic I/R as well as NLRP3 activation. In vitro, AA concentration-dependently inhibited LPS/H2O2-induced NLRP3 inflammaosome activation in KCs and RAW264.7 cells. Either GW9662 or genetic knockdown of PPARγ abolished the AA-mediated inactivation of NLRP3 inflammasome. Mechanistically, AA attenuated I/R or LPS/ H2O2-induced ROS production and phosphorylation level of JNK, p38 MAPK and IκBa but not ERK, a mechanism dependent on PPARγ. Finally, AA blocked the deleterious effects of LPS/H2O2-induced macrophage activation on hepatocyte viability in vitro, and improved survival in a lethal hepatic I/R injury model in vivo. Collectively, these data suggest that AA is effective in mitigating hepatic I/R injury through attenuation of KCs activation via PPARγ/NLRP3 inflammasome signaling pathway.
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AIM To investigate the hypothesis that treatment with dimethyl fumarate (DMF) may ameliorate liver ischemia/reperfusion injury (I/RI). METHODS Rats were divided into 3 groups: sham, control (CTL), and DMF. DMF (25 mg/kg, twice/d) was orally administered for 2 d before the procedure. The CTL and DMF rats were subjected to ischemia for 1 h and reperfusion for 2 h. The serum alanine aminotransferase (ALT) and malondialdehyde (MDA) levels, adenosine triphosphate (ATP), NO × metabolites, anti-oxidant enzyme expression level, anti-inflammatory effect, and anti-apoptotic effect were determined. RESULTS Histological tissue damage was significantly reduced in the DMF group (Suzuki scores: sham: 0 ± 0; CTL: 9.3 ± 0.5; DMF: 2.5 ± 1.2; sham vs CTL, P < 0.0001; CTL vs DMF, P < 0.0001). This effect was associated with significantly lower serum ALT (DMF 5026 ± 2305 U/L vs CTL 10592 ± 1152 U/L, P = 0.04) and MDA (DMF 18.2 ± 1.4 μmol/L vs CTL 26.0 ± 1.0 μmol/L, P = 0.0009). DMF effectively improved the ATP content (DMF 20.3 ± 0.4 nmol/mg vs CTL 18.3 ± 0.6 nmol/mg, P = 0.02), myeloperoxidase activity (DMF 7.8 ± 0.4 mU/mL vs CTL 6.0 ± 0.5 mU/mL, P = 0.01) and level of endothelial nitric oxide synthase expression (DMF 0.38 ± 0.05-fold vs 0.17 ± 0.06-fold, P = 0.02). The higher expression levels of anti-oxidant enzymes (catalase and glutamate-cysteine ligase modifier subunit and lower levels of key inflammatory mediators (nuclear factor-kappa B and cyclooxygenase-2 were confirmed in the DMF group. CONCLUSION DMF improved the liver function and the anti-oxidant and inflammation status following I/RI. Treatment with DMF could be a promising strategy in patients with liver I/RI.
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Background Caspase-1 is present in the cytosol as an inactive zymogen and requires the protein complexes named “inflammasomes” for proteolytic activation. However, it remains unclear whether the proteolytic activity of caspase-1 is confined only to the cytosol where inflammasomes are assembled to convert inactive pro-caspase-1 to active caspase-1. Methods We conducted meticulous data analysis methods on proteomic, protein interaction, protein intracellular localization, and gene expressions of 114 experimentally identified caspase-1 substrates and 38 caspase-1 interaction proteins in normal physiological conditions and in various pathologies. ResultsWe made the following important findings: (1) Caspase-1 substrates and interaction proteins are localized in various intracellular organelles including nucleus and secreted extracellularly; (2) Caspase-1 may get activated in situ in the nucleus in response to intra-nuclear danger signals; (3) Caspase-1 cleaves its substrates in exocytotic secretory pathways including exosomes to propagate inflammation to neighboring and remote cells; (4) Most of caspase-1 substrates are upregulated in coronary artery disease regardless of their subcellular localization but the majority of metabolic diseases cause no significant expression changes in caspase-1 nuclear substrates; and (5) In coronary artery disease, majority of upregulated caspase-1 extracellular substrate-related pathways are involved in induction of inflammation; and in contrast, upregulated caspase-1 nuclear substrate-related pathways are more involved in regulating cell death and chromatin regulation. Conclusions Our identification of novel caspase-1 trafficking sites, nuclear and extracellular inflammasomes, and extracellular caspase-1-based inflammation propagation model provides a list of targets for the future development of new therapeutics to treat cardiovascular diseases, inflammatory diseases, and inflammatory cancers.
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Liver ischemia/reperfusion injury (LIRI) is a common complication of liver surgery, and affects liver function post-transplantation. However, the precise mechanism underlying LIRI has not yet been completely elucidated. Previous studies have demonstrated the involvement of a number of microRNAs (miRNAs/miRs) in liver pathophysiology. The objective of the present study was to determine the potential function and mechanism of miR-101-mediated regulation of autophagy in LIRI. Compared with the sham-treated group, a significant decrease in miR-101 and mechanistic target of rapamycin (mTOR) expression levels following ischemia/reperfusion (IR) were observed, along with an increased number of autophagosomes (P<0.001). The exogenous overexpression of miR-101 has been demonstrated to inhibit autophagy during the LIRI response and the levels of mTOR and phosphorylated (p)-mTOR expression are correspondingly elevated. However, compared with the miR-NC group, miR-101 silencing was associated with reduced mTOR and p-mTOR levels and increased autophagy, as indicated by the gradual increase in the levels of the microtubule-associated protein 1 light II (LC3II). The peak levels of LC3II were observed 12 h subsequent to reperfusion, which coincided with the lowest levels of miR-101. In addition, inhibition of autophagy by 3-methyladenine significant enhanced the protective effect of miR-101 against LIRI, compared with the IR group (P<0.001). Altogether, miR-101 attenuates LIRI by inhibiting autophagy via activating the mTOR pathway.
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Hepatic steatosis renders liver more vulnerable for ischemia/reperfusion injury (IRI), which commonly occurs in transplantation, trauma, and liver resection. The underlying mechanism is not fully characterized. We aimed to clarify the role of mechanistic target of rapamycin (mTOR) signaling in hepatic ischemia/reperfusion injury (HIRI) in normal and steatotic liver using Alb-TSC1(-/-) (AT) and Alb-mTOR(-/-) (Am) transgenic mice. Steatotic liver induced by high-fat diet was more vulnerable to IRI. Activation of hepatic mTOR in AT mice decreased lipid accumulation attenuated HIRI as measured by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining, circulating levels of alanine aminotransferase and lactate dehydrogenase, and inflammatory mediators such as monocyte chemoattractant protein 1 (MCP-1), TNF-?, and IL-6 and hepatic cleaved caspase 3 in mice fed either a normal chow diet or a high-fat diet. The effects of mTOR activation on hepatic cleaved caspase 3 were reversed by rapamycin, an inhibitor of mTOR signaling. Inhibition of hepatic mTOR in Am mice increased hepatic lipid deposition and HIRI. The increment in hepatic susceptibility to IRI was significantly attenuated by pretreatment with IKK? inhibitor. Further, suppression of mTOR facilitated nuclear translocation of NF-?B p65. In conclusion, our study suggests that mTOR activity in hepatocytes decreases hepatic vulnerability to injury through a mechanism dependent on NF-?B proinflammatory cytokine signaling pathway in both normal and steatotic liver.-Li, Z., Zhang, J., Mulholland, M., Zhang, W. mTOR activation protects liver from ischemia/reperfusion-induced injury through NF-?B pathway.
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Background Ischaemia reperfusion injury is a key cause of mortality and graft loss after liver transplantation. After tissue injury, monocytes are rapidly mobilised and recruited to injured tissue by monocyte chemoattractant protein-1 (MCP-1). Elevated MCP-1 concentrations correlate with poorer outcomes in patients after haemorrhagic stroke but have not been evaluated as a prognostic marker in clinical liver transplantation. We aimed to assess the role of inflammatory monocytes and MCP-1 in ischaemia reperfusion injury Methods Adult patients undergoing liver transplantation at the Royal Free Hospital, London, UK, were recruited. Liver biopsy samples were collected preimplantation and 2 h after reperfusion from five patients. Intrahepatic mononuclear cells were extracted for immediate analysis by flow cytometery. Plasma MCP-1 concentrations from 33 patients were measured preoperatively by ELISA, 2 h and 24 h after reperfusion, and correlated with graft function by measurement of day 3 aspartate aminotransferase (AST) and early allograft dysfunction (EAD) score. Findings Flow cytometric analysis demonstrated an increase in mean classical monocytes after reperfusion compared with preimplantation (4·18% of total live cells [SD 2·61] vs 0·61 [0·38], p=0·018). In three of the five recipients we distinguished cells of donor versus recipient origin by HLA-A allele expression to demonstrate that 88% (6·24) of the classical monocytes were recipient derived in the postreperfusion biopsy sample. Median MCP-1 concentrations were significantly raised after reperfusion (385·61 pg/mL [IQR 244·75–715·20] vs 71·2 [55·61–113·99], p<0·0001) and had reduced to 740·61 (38·46–133·71) within 24 h. Patients with EAD (n=17) had significantly higher MCP-1 concentrations at 24 h than those without EAD (74·82 [66·69–219·93] vs 47·44 [29·53–77·73], p=0·037). MCP-1 concentrations at 24 h correlated with day 3 AST concentrations (p=0·002). Interpretation Our results show that classical monocytes are rapidly recruited to the liver after ischaemia reperfusion injury, and that high MCP-1 concentrations at 24 h are associated with poorer graft function. Therefore, MCP-1 blockade presents an attractive strategy to reduce graft ischaemia reperfusion injury. Funding None.