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Virus-Derived Chemokine Modulating Protein Pre-Treatment Blocks Chemokine-Glycosaminoglycan Interactions and Significantly Reduces Transplant Immune Damage

Authors:

Abstract

Immune cell invasion after the transplantation of solid organs is directed by chemokines binding to glycosaminoglycans (GAGs), creating gradients that guide immune cell infiltration. Renal transplant is the preferred treatment for end stage renal failure, but organ supply is limited and allografts are often injured during transport, surgery or by cytokine storm in deceased donors. While treatment for adaptive immune responses during rejection is excellent, treatment for early inflammatory damage is less effective. Viruses have developed highly active chemokine inhibitors as a means to evade host responses. The myxoma virus-derived M-T7 protein blocks chemokine: GAG binding. We have investigated M-T7 and also antisense (ASO) as pre-treatments to modify chemokine: GAG interactions to reduce donor organ damage. Immediate pre-treatment of donor kidneys with M-T7 to block chemokine: GAG binding significantly reduced the inflammation and scarring in subcapsular and subcutaneous allografts. Antisense to N-deacetylase N-sulfotransferase1 (ASONdst1) that modifies heparan sulfate, was less effective with immediate pre-treatment, but reduced scarring and C4d staining with donor pre-treatment for 7 days before transplantation. Grafts with conditional Ndst1 deficiency had reduced inflammation. Local inhibition of chemokine: GAG binding in donor organs immediately prior to transplant provides a new approach to reduce transplant damage and graft loss.
Citation: Zanetti, I.R.; Burgin, M.;
Zhang, L.; Yeh, S.T.; Ambadapadi, S.;
Kilbourne, J.; Yaron, J.R.; Lowe, K.M.;
Daggett-Vondras, J.; Fonseca, D.; et al.
Virus-Derived Chemokine Modulating
Protein Pre-Treatment Blocks
Chemokine–Glycosaminoglycan
Interactions and Significantly
Reduces Transplant Immune
Damage. Pathogens 2022,11, 588.
https://doi.org/10.3390/
pathogens11050588
Academic Editors: Alexander
David Barrow, Lyn Wise and
Anna Honko
Received: 18 March 2022
Accepted: 7 May 2022
Published: 16 May 2022
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4.0/).
pathogens
Article
Virus-Derived Chemokine Modulating Protein Pre-Treatment
Blocks Chemokine–Glycosaminoglycan Interactions and
Significantly Reduces Transplant Immune Damage
Isabela R. Zanetti 1, , Michelle Burgin 1, , Liqiang Zhang 1, Steve T. Yeh 2, Sriram Ambadapadi 1,
Jacquelyn Kilbourne 3, Jordan R. Yaron 1,4 , Kenneth M. Lowe 3, Juliane Daggett-Vondras 3, David Fonseca 1,
Ryan Boyd 5, Dara Wakefield 6, William Clapp 6, Efrem Lim 7, Hao Chen 8and Alexandra Lucas 1,9,*
1Center for Personalized Diagnostics (CPD), Biodesign Institute, Arizona State University (ASU),
Tempe, AZ 85287, USA; izanetti@asu.edu (I.R.Z.); mburgin@asu.edu (M.B.); liqiang.zhang@asu.edu (L.Z.);
ram.ambadapadi@asu.edu (S.A.); jyaron@asu.edu (J.R.Y.); david.fonsecaarce@yale.edu (D.F.)
2Ionis Pharmaceuticals, Inc., Carlsbad, CA 92008, USA; syeh@ionisph.com
3Department of Animal Care and Technologies, Biodesign Institute, Arizona State University (ASU),
Tempe, AZ 85287, USA; jacki.kilbourne@asu.edu (J.K.); kenneth.m.lowe@asu.edu (K.M.L.);
juliane.daggett@asu.edu (J.D.-V.)
4School for Engineering of Matter, Transport and Energy, Ira A. Fulton Schools of Engineering,
Arizona State University, Tempe, AZ 85287, USA
5Center for Applied Structural Discovery, Biodesign Institute, Arizona State University,
Tempe, AZ 85287, USA; rjboyd@asu.edu
6Pathology Department, University of Florida, Gainesville, FL 32611, USA; dwakefield@ufl.edu (D.W.);
clapp@pathology.ufl.edu (W.C.)
7The Biodesign Center of Fundamental and Applied Microbiomics, Center for Evolution and Medicine,
School of Life Sciences, Arizona State University, Tempe, AZ 85287, USA; efrem.lim@asu.edu
8The Department of Tumor Surgery, Second Hospital of Lanzhou University, Lanzhou 730030, China;
chenhao3996913@163.com
9Center for Immunotherapy, Vaccines and Virotherapy (CIVV), Biodesign Institute, Arizona State University,
Tempe, AZ 85287, USA
*Correspondence: arlucas5@asu.edu; Tel.: +1-352-672-2301
These authors contributed equally to the work.
Abstract:
Immune cell invasion after the transplantation of solid organs is directed by chemokines
binding to glycosaminoglycans (GAGs), creating gradients that guide immune cell infiltration. Renal
transplant is the preferred treatment for end stage renal failure, but organ supply is limited and
allografts are often injured during transport, surgery or by cytokine storm in deceased donors.
While treatment for adaptive immune responses during rejection is excellent, treatment for early
inflammatory damage is less effective. Viruses have developed highly active chemokine inhibitors as
a means to evade host responses. The myxoma virus-derived M-T7 protein blocks chemokine: GAG
binding. We have investigated M-T7 and also antisense (ASO) as pre-treatments to modify chemokine:
GAG interactions to reduce donor organ damage. Immediate pre-treatment of donor kidneys with
M-T7 to block chemokine: GAG binding significantly reduced the inflammation and scarring in
subcapsular and subcutaneous allografts. Antisense to N-deacetylase N-sulfotransferase1 (ASO
Ndst1
)
that modifies heparan sulfate, was less effective with immediate pre-treatment, but reduced scarring
and C4d staining with donor pre-treatment for 7 days before transplantation. Grafts with conditional
Ndst1 deficiency had reduced inflammation. Local inhibition of chemokine: GAG binding in donor
organs immediately prior to transplant provides a new approach to reduce transplant damage and
graft loss.
Keywords:
antisense; chemokine; glycosaminoglycans; inflammation; kidney; M-T7; rejection;
transplant; virus
Pathogens 2022,11, 588. https://doi.org/10.3390/pathogens11050588 https://www.mdpi.com/journal/pathogens
Pathogens 2022,11, 588 2 of 20
1. Introduction
According to the Global Observatory on Donation and Transplantation (GODT) data,
surgeries for organ transplantation in 2019 reached a record number with 153,863 trans-
plants, approximately 17.5 transplants per hour, and an increase of 4.8% from 2018 (http:
//www.transplant-observatory.org/, acessed on 1 January 2022) [
1
]. Renal transplants
comprise a large share of the transplanted organs. Diabetic and hypertensive renal dis-
ease are responsible for the majority of cases, with kidney failure requiring dialysis
and/or transplantation [
1
,
2
]. In 2018 only 36.2% of 95,479 renal transplants came from
living donors, the majority of transplants worldwide are obtained from deceased donors
(http://www.transplant-observatory.org/, accessed on 1 January 2022) and, among those,
patients with severe brain damage [
2
]. Transplanted organs are damaged even prior to
engrafting, by ischemia, low blood flow, during transport or shock, by surgical trauma
and immunological damage induced by cytokine storm in donors with severe brain in-
jury [
3
,
4
]. Thus, even before surgery, the donated organ is subjected to excess immune
cell activation and damage. This early inflammation and damage can cause long term
functional impairment [
3
5
], in addition to the acute and recurrent episodes of antibody-
mediated rejection. Early damage is linked to late progressive vasculopathy and scarring
in allografts, termed chronic rejection. Recent studies in rodent models have demonstrated
progressive inflammatory immune cell activation through cell adhesion molecule expres-
sion, causing an influx of leukocytes in the kidneys of brain-dead rats, when compared to
non-brain-dead controls [
3
,
4
]. Severe brain injury is also associated with increased cytokine
and chemokine activity, associated with immune cell activation. Increased expression
of chemokines and chemokine receptors is detected in brain death and associated with
endothelial dysfunction [36].
Viruses have developed highly effective immune modulators over millions of years of
evolution; agents designed to evade the host’s immune response to infections. We have
developed virus-derived immune-modulating proteins as a new class of therapeutic. M-T7
is a 37 KDa myxoma virus-derived purified protein that binds C, CC and CXC classes
of chemokines, blocking chemokine to GAG binding [
7
11
]. In prior studies, modifying
chemokine and chemokine receptor activity reduced transplant damage and rejection [
8
14
].
Treatment after transplant with M-T7 reduced the aortic allograft vascular inflammation
at 30 days follow-up [
8
]. M-T7 also reduced inflammation and scarring and improved
long term renal allograft survival in mouse and rat models in separate studies [
9
,
10
,
14
].
Modifying the endothelial polysaccharides and GAG composition also reduced immune
cell response and organ damage in a separate series of transplant models, including aortic
and renal allograft studies. [919].
In prior work, engrafted renal allografts reduced early histological markers of allograft
rejection with either M-T7 treatment, starting on the day of transplant, or in allograft
kidneys from mice with conditional N-deacetylase-N-sulfotransferase-1 (Ndst1) deficiency,
further supporting a central role for chemokine: GAG interaction in transplant rejection [
9
].
Ndst1 is a sulfotransferase enzyme that modifies heparan sulfate (HS), the predominate
GAG component in the endothelial glycocalyx. HS GAG binds the chemokines directing
chemokine gradient formation and cellular invasion. Donor renal allografts from Ndst1
/
donor mice had significantly reduced scores for cell infiltrates, vasculitis, glomerulitis and
tubulitis in renal allografts when compared to saline-treated control grafts [
9
]. No additional
treatment was given to these mice after transplantation, suggesting that interfering with
chemokine to HS GAG binding in donor allografts is capable of reducing early graft
inflammation, reducing immune cell activation, invasion and organ damage.
Chemokines are chemoattractant cytokines that direct immune cells to sites of injury
in donor organs and increase immune cell activation [
5
8
]. Inflammation and cellular
activation in donor organs can be protective but, when excessive, have the potential to
cause progressive damage to donor transplants with scarring, vasculitis and graft loss [
5
].
Chemokines are small proteins classified into four subgroups (C, CC, CXC and CX
3
C), as
defined by the number of amino acids (X) separating the two N terminal cysteine residues.
Pathogens 2022,11, 588 3 of 20
CC, CXC and CXC chemokines and their receptors are increased after transplant with
evidence for rejection [
5
,
6
,
20
]. Chemokines act locally to direct leukocyte adhesion, ex-
travasation and navigation along the GAG gradients in the connective tissue glycocalyx
surrounding the endothelium during immune response activation, forming gradients that
direct leukocyte traffic or invasion at sites of tissue injury [
5
,
6
]. Chemokine to receptor
interactions are promiscuous, but the combination of chemokine binding to specific recep-
tors, as well as binding to GAGs in the glycocalyx, governs the tissue immune responses.
The glycocalyx layer is composed of polysaccharides that include GAGs such as heparan
and chondroitin sulfate. Together with connective tissue proteins, the glycocalyx covers
the luminal surface of vascular endothelial cells. Interactions between chemokines and
GAGs thus allow immune cell attachment and migration [
14
,
21
,
22
] and direct immune cell
migration into engrafted organs [21,22].
The majority of current treatments for transplants are focused on decreasing immune
T cell and antibody-mediated rejection after engrafting, e.g., treatment of the transplant
recipient after surgery, to prevent rejection [
23
]. Immune cell activation and invasion, when
excessive during overactive inflammation or rejection, can damage transplanted organs
prior to transplantation, causing damage to the donor’s organs even before harvest for
transplantation, and can lead to graft loss. While current immune modulating therapeutics
are highly effective for acute antibody-mediated rejection, this treatment is less effective
for late or chronic rejection, that occurs after the first year post transplant, increasing graft
loss. Treatment for late or chronic rejection remains less effective, necessitating repeat trans-
plantation in some patients or a return to hemodialysis. There is also a risk of anti-rejection
medication induced toxicity [
23
]. High doses of immunosuppressants such as calcineurin
inhibitors and corticosteroids increase the susceptibility to opportunistic infections amongst
other severe complications, including malignancies, diabetes and Cushing’s syndrome.
New drugs are now under investigation as approaches to reduce this excess damaging
inflammation in donor grafts [2330].
Treatment of grafts prior to surgical transplant is postulated to improve graft out-
comes by decreasing inflammation and organ damage before transplantation [
10
12
,
25
34
].
Dopamine pre-treatment of donor organs has demonstrated reduced renal allograft rejection
and superior long-term graft survival [
24
]. Similar beneficial findings have been observed
with pre-treatment of brain-dead donor vascular composite allografts (VCAs) with CR2-
Crry, a targeted complement inhibitor, that blocks C3 activation and the generation of
biologically active complement, specifically C3 opsonins, C3a, C5a and the membrane
attack complex. C3a and C5a are involved in endothelial activation and immune cell
recruitment [
31
]. Pre-coating of grafts with CR2-Crry ameliorated the ischemia-reperfusion
injury (IRI) in grafts from brain-dead donors and prolonged graft survival in animal mod-
els [
31
]. Recent studies have investigated pre-treatment of liver and heart transplants with
small interfering RNA (siRNA) approaches with different targets, including caspase 3 and
complement factor C5a, in preclinical models [32,33].
In summary, chemokine: GAG interactions are predicted to drive damaging immune
cell invasion in engrafted organs. We have postulated that treatment of renal allografts prior
to engrafting, e.g. pre-treatment with therapeutics designed to interfere with chemokine:
GAG interactions, will reduce early donor graft damage and rejection (Figure 1). Here we
investigate pre-treatment (PT) with both M-T7, as well as an antisense (ASO) construct
targeting Ndst1 (ASO
Ndst1
) prior to donor organ implantation. Pretreatment of donor
kidneys was given either immediately prior to engrafting with soaking for 1 h before
subcapsular allograft implant (PTS), or by pre-treatments given for 7 days to the donor
mouse before organ resection and transplant (7dsPT) (Figure 2). Subcapsular renal as
well as subcutaneous transplants were both examined after PTS (soaking) pre-treatment.
Our studies indicate that interfering with the chemokine: GAG axis immediately prior to
transplantation can reduce early transplant immune damage.
Pathogens 2022,11, 588 4 of 20
Figure 1. Chemokine GAG interaction–M-T7 and ASONdst1 .
Figure 2. Flow Diagram of Transplant Studies.
2. Results
2.1. Soaking Pre-Treatment (PTS) of Subcapsular Renal Allografts with M-T7 Significantly
Reduced Inflammation at 15 Days Follow Up
At 15 days follow-up, the areas and diameters of inflammation in the engrafted
sections pretreated by soaking with M-T7, were significantly reduced when compared to
Saline PTS (Figure 3A–E; p< 0.0003). Micrographs of renal allograft implants at 15 days are
shown in Figure 3, panels A–D. An independent histopathology score analyzing tubulitis,
glomerulitis, scarring and a combined histopathology score detected similar significant
reductions with M-T7 treatment at 15 days follow-up in grafts pre-treated for one hour by
PTS (Figure 3, Panels G,H) immediately prior to transplantation.
Renal allograft subcapsular implants pretreated with M-T7 by soaking (PTS) for one
hour prior to subcapsular transplant produced a trend toward a decrease in inflammation at
3 days follow-up, but this trend did not reach significance (Figure 3E; p= NS). Histopathol-
ogy scores for glomerulitis (Figure 3G, p< 0.0188 ANOVA) and the overall histopathology
score (Figure 3H, p< 0.0002 ANOVA) were significantly reduced after M-T7 PTS at 15 days.
ASO
Ndst1
did reduce inflammation at 3 and 15 days in comparison to saline (p< 0.0001
ANOVA; p< 0.0403 for 3 days follow-up and p<0.0003 at 15 days follow-up). However,
Pathogens 2022,11, 588 5 of 20
the ASO
Ndst1
effects at 15 days were equal to the control ASO
Scr
(p= 0.9763) suggesting a
non-specific effect for ASONdst1 on inflammation in PTS treated grafts (Figure 3D,F).
Figure 3.
Inflammation in PTS pretreatment groups
.
Reduced inflammation is detected on H&E
stained micrographs with M-T7 PTS pre-treatment of donor organs given immediately prior to
engrafting. Black arrows indicate inflammatory cell invasion, red arrows highlight areas of scarring.
(
A
). Subcapsular renal allografts pretreated with saline at 15 days follow-up, 20
×
; (
B
). Subcapsular
renal allograft pretreated (PTS) with saline at 15 days follow-up, 40
×
(
B
); (
C
). PTS with M-T7
at 15 days follow-up showing decreased inflammation in comparison to saline, 40
×
; (
D
). (PTS)
with ASO
Ndst1
at 15 days follow-up, 40
×
. Black arrows indicating inflammation; (
E
). Bar graphs
comparing ratios of mean area of inflammation/ total renal allograft area for saline versus M-T7
PTS allografts at 3 and 15 days. M-T7 significantly decreased area of inflammation in comparison
to saline at 15 days follow-up (*** p< 0.0001 ANOVA, *** p< 0.0003); (
F
). Bar graphs comparing
ratios of mean area of inflammation/total renal allograft area days (*** p< 0.0001 ANOVA; p< 0.0403)
and at 15 days (*** p< 0.0003) in comparison to saline. ASO
Ndst1
effects were equal to the control
ASO
Scr (p= 0.9763)
suggesting a non-specific effect for ASO
Ndst1
on inflammation in PTS treated
grafts; (
G
). Bar graphs comparing glomerulitis histopathology score of M-T7 PTS renal allografts
versus controls. Glomerulitis score was significantly reduced with M-T7 treatment (*** p< 0.0188
ANOVA) at 15 days follow-up; (
H
). Bar graphs showing decreased combined histopathology score
with M-T7 PTS renal allografts at 15 days follow-up in comparison to controls (*** p< 0.0002 ANOVA).
*p< 0.05, *** p< 0.001.
At 3 and 15 days follow-up, a reduction in the measured area for scarring in allografts
was detected with the M-T7 PTS treatment. M-T7 reduced scarring both as a measured area
(Figure 4A,B) and as a histopathology score (Figure 4C, ANOVA p< 0.0302). Tubulitis scores
were not significantly changed by any treatment (p= NS, not shown). ASO
Ndst1
reduced
measured inflammation at 15 days follow-up in the PTS model (p< 0.0403, Figure 3F)
but did not have a detected significant reduction on pathology scoring for glomerulitis
(Figure 3G) or combined score (Figure 4C). ASO
Ndst1
PTS treatment did demonstrate a de-
crease in histopathology score for scarring (p< 0.0425, Figure 4C), but did not demonstrate
a significant decrease in measured scar areas (Figure 4A). A trend towards an increased
number of detected glomeruli with intact morphometry was seen in M-T7 PTS treatments,
but did not reach significance (Figure 4D, ANOVA p< 0.5437; p= NS).
Pathogens 2022,11, 588 6 of 20
Figure 4.
Scarring in PTS pretreatment groups. Analysis of scarring in PTS groups. (
A
). Bar graphs
for mean area of Scarring/Total allograft Area at Subcapsular Renal allografts pre-treated (PTS) with
MT7 in comparison to Saline. Reduction in the area of scarring was detected at both 3 and 15 days
follow-up (p < 0.0001 ANOVA; *** p< 0.0003) with M-T7 treatment; (
B
). Bar graphs for Ratio of
mean Area of Scarring/Total Allograft Area at Subcapsular Renal Allografts pre-treated (PTS) with
ASO
Ndst1
. No significant decrease was found for measured scar area at both 3 and 15 days follow-up;
(
C
). Bar graphs for independent Histopathology Scar Score at Subcapsular Renal Allografts PTS at
15 days follow-up. M-T7 has significantly reduced area of scarring (p< 0.0302 ANOVA). ASO
Ndst1
has also demonstrated reduction on histopathology score for scarring (p< 0.0425); (
D
). Bar graphs for
number of Detected Glomeruli/Total Graft area with PTS of Subcapsular Renal Allografts at 15 days
follow-up. A trend toward an increased number of detected glomeruli with intact morphometry was
found with M-T7 PTS treatments but significance was not reached (p< 0.5437 ANOVA). * p< 0.05,
*** p< 0.001.
Implant of kidney allografts derived from conditional Ndst1 knockout (Ndst1
/
)
mice also significantly reduced inflammation and scarring at 3 days post-transplant
(Figure 3F,
p< 0.0403). These findings are consistent with prior work where transplant of
whole functioning Ndst1/transplants (renal allografts derived from conditional Ndst1
knock out mice) into BALB/c mice produced a significant reduction in acute rejection [9].
ASO
Ndst1
PTS reduced the independent histopathology scores for scarring (Figure 4C,
p< 0.0425), but neither for glomerulitis (p= 0.6820) nor for the overall histopathology
score (p= 0.0689), when compared to saline or ASOScr control PTS treatments (Figure 3G).
For glomerulitis, ASO
Scr
treatment was similar to saline treatment, suggesting that this
histopathology scoring assessment also demonstrated a nonspecific effect for ASO
Ndst1
and
ASOScr treatments.
No adverse effects were detected with either treatment approach, M-T7 or ASO
Ndst1
,
with neither increased mortality nor infections, as seen in prior work [
7
9
,
14
,
35
,
36
]. Overall,
the mortality for subcapsular transplants was 80%, with no significant differences for any
of the treatments, whether given as PTS or as 7ds PT.
2.2. Immunohistochemical Analyses of Immune Cell Invasion
The only treatment given to allografts in the PTS or 7ds PT models was given prior
to engrafting, with no follow-up treatment of recipient mice after engrafting. Early effects
on immune cell responses were therefore assessed at 3 days after transplantation. F4/80+
Macrophage infiltrates were significantly reduced by M-T7 treatment at 3 days follow-up
in the subcapsular PTS model (ANOVA p< 0.0008) (Figure 5A).
Pathogens 2022,11, 588 7 of 20
Figure 5.
Immunohistochemistry demonstrates M-T7 reduces macrophage invasion in the immediate
pre-treated (PTS) model. (
A
). F4/80+ Macrophage infiltrates were significantly reduced with M-T7 at
3 days follow-up (p< 0.0008 ANOVA). Reductions in this model were comparable to the ones seen in
the Ndst1
/
kidney transplants when compared to saline (p< 0.0009). ASO
Ndst1
, in contrast, did
not alter macrophage invasion (p= 0.2007); (
B
). CD3+ T cell counts were not significantly altered by
any of the pre-treatments at 3 days follow-up (p= 0.1944 ANOVA); (
C
). LyG6+ neutrophil cell counts
at 3 days follow-up were not significantly altered by pre-treatments (p= 0.0684 ANOVA). Ndst1
/
subcapsular transplants had reduced neutrophil counts when compared to saline, while M-T7 and
ASO
Ndst1
trended toward a non-significant increase; (
D
). CD19+ B cell counts were significantly
decreased (p< 0.0164 ANOVA) by ASO
Ndst1
(p< 0.0131) and Ndst1
/
(p< 0.0400) pre-treatment
of C57BL/6 mice at 3 days follow-up. M-T7 and saline treatments had equivalent effects on CD19+
B cell counts indicating neither suppression of nor increase in B cells by M-T7. Micrographs of pre-
treated (PTS) subcapsular renal allografts with: (
E
). Saline PTS at 3 days follow-up showing F4/80+
macrophages, 100
×
; (
F
). M-T7 PTS at 3 days follow-up showing decreased F4/80+ macrophages,
100
×
. Glomeruli visualized inside the transplant; (
G
). Saline PTS at 3 days follow-up showing CD3+
T cells, 100
×
; (
H
). CD3+ T cells in M-T7 PTS at 3 days follow-up, 100
×
; (
I
). IHC for CD19+ B cells in
Saline PTS at 3 days follow-up, 100
×
; (
J
). IHC with CD19+ B cells in M-T7 PTS at 3 days follow-up,
histology micrograph at 100×shows intact glomeruli.
M-T7 PTS soaking significantly reduced the macrophage invasion (p< 0.0006), with
reductions similar to the reductions seen in Ndst1
/
conditional knock out kidney trans-
plants when compared to Saline PTS (p< 0.0009) (Figure 5A,E,F). In contrast, ASO
Ndst1
did
not alter macrophage invasion (p= 0.2007). CD3 positive (CD3+) T cell (Figure 5B,G,H;
p= 0.1994) and Ly6G+ neutrophil counts (Figure 5C; p= 0.0684) in each graft implant
were not significantly altered by any of the pre-treatments. Ndst1
/
implants at 3 days
follow-up had reduced neutrophil counts when compared to Saline PTS treatment of WT
C57BL/6 kidney implants, while M-T7 and ASO
Ndst1
trended toward a non-significant
increase (Figure 5C). CD19 B cell counts were significantly decreased (ANOVA p< 0.0164)
by the ASO
Ndst1
pre-treatment of C57BL/6 mice (p< 0.0131) and in the Ndst1
/
allograft
implants (p< 0.0400) (Figure 5D). M-T7 and saline treatments had equivalent effects on the
CD19 B cell counts indicating that M-T7 produced neither detrimental suppression of, nor
an increase in, B cells, (Figure 5D,I,J). These observations suggest that M-T7 predominantly
reduced macrophage invasion in the immediate pre-treatment PTS model.
Pathogens 2022,11, 588 8 of 20
2.3. Seven Day Pretreatment (7dsPT) with M-T7 or ASONdst1 Did Not Reduce Inflammation,
but Did Reduce Scarring
Seven days of pre-treatment of donor mice with either ASO
Scr
or ASO
Ndst1
increased
the areas of inflammatory cell invasion when compared to Saline 7dsPT at 3 days follow-up
(Figure 6A; p< 0.0044 ANOVA). M-T7 7dsPT did not reduce inflammation when compared
to Saline 7dsPT at both 3 and 15 days follow-up (Figure 6A,B,G,H). At 15 days follow-up,
there was no significant change in the area of inflammation with any treatment, although
there was a trend toward increased inflammation with ASO
Ndst1
(ANOVA
p= 0.2771)
(Figure 6B).
ASO
Scr
7dsPT at 3 days follow-up led to increased inflammation when com-
pared to M-T7 (p< 0.0026) but not in comparison to ASO
Ndst1
7dsPT
(p= 0.1433).
Scarring
was significantly reduced by the pre-treatment of donor mice with M-T7
(p< 0.0010),
and by ASO
Ndst1
7dsPT (p= 0.13144) at 3 days follow-up, when compared to saline or
ASO
Scr
controls (Figure 6C, ANOVA p< 0.0033). At 15 days follow-up, there was again
no overall decrease with 7dsPT with either ASO
Ndst1
(p= 0.3690) or M-T7 (p= 0.4517)
when compared to saline pre-treatments of donor mice for 7 days prior to transplant
(p< 0.0556,
ANOVA). ASO
Scr
treatment did increase scarring and was significantly greater
than ASONdst1 (p< 0.0479) when given as 7dsPT at 15 days follow-up.
Figure 6.
Inflammation and scarring after 7dsPT. Seven days pre-treatment of donor mice with M-T7
did not reduce inflammation, but reduced scarring. (
A
). Bar graphs of 7 days pre-treatment of Subcap-
sular Renal Allografts comparing Mean area of inflammation/Total allograft area at 3 days follow-up.
ASO
Scr
and ASO
Ndst1
increased areas of inflammation when compared to Saline
(p< 0.0044
ANOVA).
M-T7 did not reduce inflammation when compared to saline; (
B
). Bar graphs of 7 days pre-treatment
of Subcapsular Renal Allografts comparing Mean area of inflammation/Total allograft area at 15 days
follow-up. There was a trend towards increased inflammation with ASO
Ndst1
(p= 0.2771 ANOVA),
but no significant change with any treatment; (
C
). Bar graphs for Mean area of Scar/Total Renal
Allograft area at 3 days follow-up (p< 0.0033 ANOVA). Scarring was significantly reduced by 7dsPT
with M-T7 (p< 0.0010), and by ASO
Ndst1
7dsPT (p= 0.13144), when compared to saline or ASO
Scr
controls; (D). Bar graphs for Mean area of Scar/Total Renal Allograft area at 15 days follow-up
Pathogens 2022,11, 588 9 of 20
(p< 0.0556
ANOVA). There was no overall decrease with 7dsPT with either ASO
Ndst1
(p= 0.3690)
or M-T7 (p= 0.4517) treatments when compared to saline. ASO
Scr
treatment significantly increased
scarring in comparison to ASO
Ndst1
(p< 0.0479); (
E
). Bar graphs for F4/80 macrophages cell count of
7dsPT of Subcapsular Renal allograft at 3 days follow-up. M-T7 and ASO
Ndst1
had nonsignificant
trends toward reducing F4/80+ cell counts (p= 0.1434 ANOVA); (
F
). Bar graphs for CD3+ T cell count
of 7dsPT of Subcapsular Renal allograft at 3 days follow-up. ASO
Scr
increased CD3+ T cell counts
when compared to Saline, M-T7 or ASO
Ndst1
pre-treatments (p= 0.0135 ANOVA). (
G
). H&E Histology
micrograph of Subcapsular Renal Allograft after 7dsPT with ASO
Scr
at 15 days follow-up showing
increased area of scarring, 40
×
; (
H
). H&E Histology micrograph of Subcapsular Renal Allograft after
7dsPT with M-T7 at 15 days follow-up showing decreased area of scarring, 40×. ** p< 0.01.
CD3+ T cell and F4/80 macrophage counts (Figure 6E,F) had only borderline de-
creases on IHC analysis, paralleling the lack of marked efficacy in reducing inflammatory
cell invasion when donor mice were pretreated for 7 days prior to graft harvesting and
transplant, in contrast to the soaking PTS approach. M-T7 and ASO
Ndst1
had nonsignificant
trends toward reducing F4/80+ cell counts (p= 0.1434, Figure 6E) with trends toward
reduced macrophage counts for M-T7, ASO
Ndst1
as well as ASO
Scr
treatments. ASO
Scr
pre-treatment increased CD3+ T cell counts when compared to Saline, M-T7 or ASO
Ndst1
pre-treatments (ANOVA p= 0.0135, Figure 6F). M-T7 and ASO
Ndst1
did not significantly
increase or decrease F4/80 macrophage or CD3 positive T cells with 7dsPT when compared
to saline (Figure 6E,F).
These findings would suggest that immediate pre-treatment with M-T7 for 1 h prior to
allograft transplant was more effective than pre-treatment for 7 days (7dsPT) for preventing
inflammation or scarring in renal allografts.
2.4. C4d Positive Staining Is Increased in Both the Subcapsular Allografts as Well as the
Recipient Kidney
As a secondary analysis for rejection, immunohistochemical staining was performed
for the detection of C4d (complement). Areas of dense C4d positive tubules were detected
in both the renal allograft implants and in the recipient kidneys with both the PTS and
7dsPT approaches (Figure 7). While subcapsular renal allografts pre-treated for one hour
(PTS) with M-T7 before transplantation had significantly decreased inflammation at 15 days
follow-up (p< 0.0066) (Figure 3E), a reduction in scarring was detected in the 7dsPT model
(p< 0.0033 ANOVA) at 3 days follow-up with M-T7 pre-treatment (Figure 6C), but not at
15 days. C4d staining in the PTS model did not show a significant reduction.
A nonsignificant trend was detected for decreased inflammation in the renal grafts
with M-T7 and ASO
Ndst1
treatment in the 7dsPT model, but scarring was reduced with
both treatments at 3 days follow-up (Figure 6C). In contrast, the implanted allograft kid-
ney had significantly decreased C4d positive staining (p< 0.0001) at 15 days follow-up
(Figure 7A,E,F,G),
with either M-T7 (p< 0.0201) or ASO
Ndst1
(p< 0.0001) pre-treatments for
7 days.
ASO
Ndst1
and M-T7 counts were reduced compared to both ASO
Scr
and Saline treat-
ment controls. Thus, ASO
Ndst1
may be more beneficial when given for 7 days to the donor
prior to transplantation rather than when given as a soaking pre-treatment for 1 h. Implant
of the Ndst1/conditional knockout graft was conversely less effective (not shown).
There was an increase in detectable C4d positive staining in the recipient BALB/c
mouse kidney (Figure 7B,D), as well as in the C57Bl/6 allograft (Figure 7D,E) suggesting a
possible systemic response after transplant, either induced by surgical injury or rejection.
This recipient kidney increase in C4d staining was not altered by either PTS or 7dsPT for
any treatments (Figure 7B).
These findings indicate that 7 days pre-treatment (7dsPT) of donor animals may be a
more effective approach for reducing the adaptive immune responses, while immediate
pre-treatment soaking may be better when treating with local M-T7 inhibition of chemokine:
GAG interactions and inflammation. Further, the allograft implant induces a widespread
Pathogens 2022,11, 588 10 of 20
renal and/or systemic response and this response is suppressed by M-T7 PTS or 7ds PT
soaking pre-treatments.
Figure 7.
Immunohistochemical analysis C4d staining Allografts and Recipients- 7dsPT and PTS.
Immunohistochemical analysis of C4d within Subcapsular Renal Allografts and the recipient Kidney
with both 7dsPT and PT approaches. Areas of dense C4d positive tubules were detected in both
models. (
A
). Bar graphs for C4d positive cell counts inside renal allograft sections with 7 days pre-
treatment at 15 days follow-up (p< 0.0001 ANOVA). Both M-T7 (p< 0.0201) and ASO
Ndst1 (p< 0.0001)
significantly decreased C4d positive staining in comparison to ASO
Scr
and Saline treatment controls;
(
B
). Bar graphs for C4d positive cell count in the recipient kidney pretreated for 7 days at 15 days
follow-up. C4d staining was not altered by any treatment (p= 0.3263 ANOVA); (
C
). Bar graphs for
C4d positive cell count at Subcapsular Renal Allograft with PTS at 15 days follow-up. Increase in C4d
staining was not altered by any of the treatments (p= 0.5597 ANOVA); (
D
). Histology micrograph of
Subcapsular Renal allograft and Recipient kidney with immunohistochemistry pretreated for 7 days
with saline at 15 days follow-up, 4
×
. Small black arrows point to areas of dense C4d positive staining;
big black arrows point to inflammatory cells. Histology micrographs of subcapsular renal allograft
pretreated for 7 days at 15 days follow-up (7dsPT) with: (
E
). saline. IHC showing C4d positive cells,
100×; (F). ASOScr, 100×; (G). M-T7, 100×.
2.5. Subcutaneous Renal Allograft Transplant Demonstrated a Significant Reduction in
Inflammation and Scarring with M-T7 PTS
To rule out nonspecific effects secondary to the immediate soaking pre-treatment
(PTS) with M-T7, we repeated the study in a second allograft transplant model using a
subcutaneous renal allograft transplant model. Sections of kidney from C57Bl/6 mice
were soaked for one hour with either M-T7, ASO
Ndst1
, ASO
Scr
or Saline, as for the PTS
for subcapsular transplants, and then implanted as subcutaneous transplants. No further
treatment was given after engrafting. PTS with M-T7 again significantly reduced the
inflammation in renal allograft implants, as seen with the renal subcapsular transplant
model (ANOVA p< 0.0088; M-T7 p< 0.0026; Figure 8A,D,E). Thus, there was reproducible
efficacy in a second allograft implant.
Pathogens 2022,11, 588 11 of 20
Figure 8.
Subcutaneous Allograft Transplant–PTS treatment pre-engrafting. Analysis of Results
for the pre-treatment (PTS) of Subcutaneous Renal Allograft Transplant Model. Pretreatment with
M-T7 has significantly decreased inflammation and scarring. (
A
). Bar graphs comparing ratio of
inflammation diameter/total graft diameter for the different pre-treatments of subcutaneous allograft
at 15 days follow-up. PTS with M-T7 significantly reduced inflammation (M-T7 p< 0.0001,
p< 0.0088
ANOVA). Pretreatment in Ndst1
/
deficient mouse renal transplants significantly reduced inflam-
mation; (
B
). Bar graphs comparing ratio of scarring diameter/total graft diameter for the different
pre-treatments of Subcutaneous allograft at 15 days follow. None of the treatments reduced scar-
ring in the subcutaneous transplant model (p= 0.0567 ANOVA); (
C
). Bar graphs comparing F4/80+
macrophage cell count for the different pre-treatments of subcutaneous allografts at 15 days follow-up
(p = 0.0012 ANOVA). ASO
Ndst1
has significantly decreased macrophage count in comparison to saline
and ASO
Scr
(p< 0.0079). F4/80+ Cell count was also decreased in Ndst1 deficient mouse renal
transplants (p< 0.0026); (
D
). H&E Histology micrograph of Subcutaneous Renal Allograft pretreated
(PTS) with ASO
Scr
at 15 days follow-up. Yellow arrows pointing to area of dense inflammation at
the transplant, 20
×
; (
E
). H&E Histology micrograph of Subcutaneous Renal Allograft pretreated
(PTS) with M-T7 at 3 days follow-up. Yellow arrows pointing to inflammatory cells, 20
×
. ** p< 0.01,
*** p< 0.001.
ASO
Ndst1
PTS treatment neither reduced inflammation nor scarring and did not sig-
nificantly differ from ASO
Scr
(Figure 8A,B), again indicating a nonspecific effect
(p= 0.096
for ASO
Ndst1
;p< 0.002 for ASO
Scr
). Ndst1
/
deficient mouse renal transplants did sig-
nificantly reduce inflammation as seen in the subcapsular transplant model (Figure 8A;
p< 0.0026).
In contrast to the subcapsular transplant model, none of the treatments significantly
reduced scarring after subcutaneous transplant (p= 0.0567 ANOVA; Figure 8B), although
there was a trend. This difference in the effect on scarring may be due to the reduced
vascularity in the dermal implant sites or due to the marked differences in the local intrinsic
immune responses in the dermal layers when compared to the kidney capsule, which may
or may not be inter-related.
Pathogens 2022,11, 588 12 of 20
3. Discussion
Chemokine: GAG interactions are central to immune cell responses, providing a
directional map or gradient for immune cellular invasion at sites of injury [
37
]. This integral
chemokine response is a local response initiating immune cell responses at sites of tissue
injury. We have assessed the efficacy of local disruption of chemokine: GAG interactions in
donor organs immediately prior to transplant as a new therapeutic approach to interrupting
immune responses and inflammation in donor organs. We have examined the blockade of
chemokine to GAG binding using both the Myxoma virus-derived chemokine modulating
protein, M-T7, as well as modifying the Heparan sulfate GAG composition using ASO
Ndst1
treatments. We have detected a significant reduction in inflammation after treating donor
organs immediately prior to transplant with M-T7, a chemokine-modulating protein that
blocks chemokine: GAG interactions. Immediate pre-treatment of the donor organ with
M-T7 had greater efficacy than systemic pre-treatment of donors. In contrast, treatment
with ASO
Ndst1
was not effective when given immediately prior to transplantation, but
did show efficacy when given to the donor mouse for 7 days prior to transplant, where
M-T7 was not as effective. In summary, interruption of chemokines to GAG binding with
M-T7 was effective when given immediately prior to transplant, whereas modification of
HS GAG composition with ASO
Ndst1
required prolonged treatments of the donor prior
to engrafting.
In prior work we demonstrated that treatment beginning after transplant, using either
the chemokine modulating protein M-T7 that blocks chemokine: GAG interactions or the
engrafting of Ndst1 deficient (Ndst1
/
) mouse donor kidneys, led to significant reductions
in early and late rejection [
9
]. Here, pre-treatment with either M-T7 or ASO
Ndst1
in donor
allografts was assessed as an approach to prevent early damage and ongoing severe
inflammation in donor organs. We examined both initial soaking for one hour immediately
prior to transplantation and treatment with systemic injections of donor animals for 7 days
prior to transplant. As noted, there was a greater benefit with reduced inflammation and
scarring (fibrosis) after M-T7 treatment with the immediate pretransplant soaking (PTS),
rather than the 7dsPT. M-T7 also provided a greater reduction in inflammation than did
ASO
Ndst1
. Given the nature of the chemokine to GAG interaction, which is presumed to be
a local GAG to chemokine binding, this would appear to emphasize the central roles of
early local chemokine GAG gradients in donor organs in driving immune cell binding and
invasion after transplantation.
As a second analysis for pre-treatment, the donor mouse was treated for 7 days prior
to harvest of the kidney for transplantation. In these 7dsPT donor organs there was no
significant reduction in inflammation, but there was a significant reduction at 3 days follow-
up. Both the M-T7 and ASO
Ndst1
significantly reduced scarring in the grafts (Figure 5) and
both were reduced compared to ASOScr control and saline controls. Interestingly, the C4d
marker for rejection was reduced by ASO
Ndst1
7ds PT treatment of donors, while M-T7 was
not effective. C4d staining was detected in the recipient kidneys remote from the engrafted
organs, but there was no significant change with any treatment for this detected C4d marker
in the recipient kidneys. The cause for this detected C4d may be simple organ damage
from the surgical implant or true rejection, and may represent a systemic response or rather
a local injury response via vascular or lymphatic interactions between the graft site and the
recipient kidney. The subcapsular and subcutaneous transplants are predicted to have a
significant ischemic component, in addition to the expected incompatibility due to mouse
strain mismatch (BALB/c and C57BL/6). This will require further study to demonstrate
the cause for this recipient reaction.
Prior work demonstrated efficacy in renal and aortic allografts with M-T7 treatment
given after transplant by iv or ip injections [
9
,
14
]. This efficacy may, however, be due to
both the local and systemic immune modulating effects of M-T7 treatments. We have now
demonstrated that modulation of chemokine: GAG interactions using M-T7 immediately
prior to graft implant, employing either the soaking or PTS method, is superior to pre-
treatment of donor animals for 7 days prior to transplantation for reducing inflammation
Pathogens 2022,11, 588 13 of 20
and rejection. We would postulate that the local pre-treatment or soaking PTS approach
provides an immediate directed treatment acting on the donor organ. With pre-treatment of
the donor mouse for 7 days, the reduced efficacy, if any, was detected in the Subcapsular PTS
model. This may be due to lack of effective access of the differing treatments to the donor
organ or more rapid clearing. ASO
Ndst1
treatment did demonstrate reductions in detectable
Ndst1 expression in normal mice. We do not, however, know the treatment distribution
of M-T7 in the donor mice treated systemically. ASONdst1 also reduced scarring as well as
C4d staining when given to the donor mouse for 7 days prior to transplant. ASONdst1
treatment may potentially require longer term application for efficacy or may simply not
modify the HS GAG glycocalyx composition in a manner that would effectively reduce
immune cell invasion. Future work with functional allograft transplants will be a next
stage to assessing pre-treatment with agents that modify chemokine: GAG interactions.
The implant of the Ndst1
/
mouse kidneys, derived from conditional knock out
mice, when transplanted using the subcapsular PTS model approaches did demonstrate
significant reductions in inflammation as well as scarring in the subcapsular PTS model.
This efficacy of the Ndst1
/
conditional KO mouse implant for reducing early inflam-
mation and scarring is consistent with an intrinsic benefit for reducing Ndst1 expression
in the endothelial cell and myeloid precursor cell populations. This benefit did not, how-
ever, translate fully when treating the mouse donor kidneys with PTS or systemic 7ds
PT approaches. This reduced benefit with ASO
Ndst1
pre-treatments may reflect the fact
that systemic ASO treatment did not alter graft GAG composition in the same manner as
selective endothelial cell conditional knock-out in Ndts1
/
mouse model. The chemokine
to GAG interaction is thus a complex multifaceted interaction dependent upon an array
of differing chemokines, receptors and GAG species. There is redundancy and overlap of
many chemokine interactions with differing cell types. Thus, a non-specific Ndst1 sup-
pression of expression may be less effective or may require longer term treatments after
transplantation. Newer ASO constructs are in development by Dr S Yeh and may prove to
have greater efficacy.
With each model we examined pre-treatment alone. Neither subsequent immune
modulating or immunosuppressant treatment, nor any M-T7 or ASO
Ndts1
treatment, were
given after transplantation. In future work, additional treatments could be given after
the initial pre-treatment, as well as potential combined treatment with standard immune
suppressants used to prevent rejection. In addition, it should be noted that these are
useful models to complete an initial screening for the benefit of pre-treatments, but an
analysis of pre-treatment efficacy with and without subsequent ongoing treatments after
transplantation should be examined for further efficacy.
To investigate the potential role of modulating chemokine: GAG interactions to re-
duce renal allograft transplantation we also analyzed two alternative models for testing
therapeutics. We have examined both subcutaneous and subcapsular implants as models
for renal transplant rejection. Here we demonstrated that both transplant models, kidney
to kidney subcapsular and kidney to subcutaneous transplantation, detected significant
changes in the generalized inflammatory cell invasion, but scarring was more improved in
the subcapsular transplant model after treatment with M-T7. The subcapsular model may
provide a better model for transplant rejection than subcutaneous kidney transplantation
and provides a closer physiological equivalent to full functional kidney transplant. The two
differing recipient mouse implant areas, subcapsular kidney and subcutaneous implants,
have known differences in vascularization as well as in local immune responses.
Subcutaneous transplantation has been studied using different types of organ tissue
including trachea and hepatocytes [
38
40
], however, although it constitutes an easy and
accessible method for surgery, the subcutaneous area is less vascularized than other trans-
plantation sites [
41
]. What may be of greater relevance is that the skin has a very unique and
active immune system differing extensively from many of the internal organs including
the kidney [
42
], which constitutes a great limitation for the success of graft transplant.
Studies using subcapsular kidney transplants have presented good results with different
Pathogens 2022,11, 588 14 of 20
organ tissues, including cornea and hepatocytes, the latter with better outcomes specifically
in ischemia-reperfusion injury [
43
,
44
], contrasting with results in subcutaneous models.
Furthermore, the recipient’s microenvironment under the renal capsule is believed to be
much more suitable to receive organ tissue than the skin; this supposition is corroborated
by results from studies with transplantation of kidney organoids under the kidney capsule
resulting in formation of glomerular basement membrane, fenestrated endothelial cells
and podocyte foot processes in the absence of any exogenous vascular growth factor [
45
].
These studies indicate that the use of subcapsular renal transplants provides a simpler
allograft transplant model for the study of transplant rejection and vasculopathy as well as
new treatment approaches. Full orthotopic kidney transplants in mice requires extensive
training in microsurgery, as well as a large number of mice, and the surgeries are techni-
cally complex due to their small size. These alternative and simpler approaches in animal
models using subcapsular and subcutaneous transplants may offer similar benefits in the
evaluation of inflammation and response to treatment, while having lower costs, surgical
preparation time and quantity of surgical supplies [46].
Virus-derived immune modulating proteins protect the virus during infection, block-
ing host immune defenses, especially immune cell invasion. The virus-derived chemokine
modulating protein M-T7 reduced renal allograft inflammation and scarring in subcapsular
and subcutaneous transplants when given to the donor organ as a pre-treatment immedi-
ately prior to transplant. Viruses provide a new approach for investigating mechanisms of
transplanted organ rejection and a potential source of new immune modulating protein
therapeutics for prevention of transplant organ damage and rejection.
4. Methods
4.1. Pretreatment of Allografts: Pretreatment Soaking (PTS) of Renal Allografts and 7 Days
Pretreatment of Renal Allograft Donors (7dsPT)
All renal allograft procedures were approved by the ASU Institutional Animal Care
and Use Committee (IACUC) and conformed to national, international and university
guidelines for animal care. We have examined two approaches to pre-treatment of allografts
and two approaches for the blockade of chemokine: GAG interactions (Figure 2). Treatment
with either M-T7, a chemokine-modulating protein (CMP) that interferes with chemokine:
GAG binding as well as pre-treatment with ASO
Ndst1
were assessed (Table 1, Figure 1). Both
soaking pre-treatment/PTS (N = 50 transplants) and 7 days donor pre-treatment/7dsPT
(N = 48 transplants) approaches were investigated using treatment with either M-T7 or
ASO
Ndst1
in the subcapsular transplant model. For subcutaneous transplant, only the PTS
pre-treatment was examined (N = 62 transplants). As comparators, saline-treated allografts,
scrambled sequence ASO (ASO
Scr
) controls or kidneys isolated from a genetic knock out of
Ndst1 (Ndst1/) were assessed in renal allograft implants.
For soaking pre-treatments, PTS, given immediately before transplantation, kidneys
from C57BL/6 donor mice after euthanasia were flushed by hand using sterile technique
with 200
µ
L of either 1
µ
g/mL M-T7, 25 mg ASO
Ndst1
, 25 mg ASO
Scr
or saline controls
and then placed in oxygenated Dulbecco’s Modified Eagle Medium (DMEM), for one
hour prior to transplantation (Figure 2) [
32
]. For systemic pre-treatment of the donor
mice, C57BL6 mice were given intraperitoneal (IP) injections daily for 7 days with 200
µ
L
of either M-T7 protein (1 ng/g body weight) diluted in sterile saline or 25 ng/gm body
weight ASO
Ndst1
, or controls, specifically 25 ng/gm ASO
Scr
or Saline. Ndst1
/
renal
transplants also served as a non-treated control where recipient BALB/c mice received
kidney transplants from mice conditionally deficient in Ndst1 [
27
]. Renal allografts were
treated by PTS for subcapsular and subcutaneous allograft implants. In a second cohort,
allografts were also pretreated by giving 7 days pre-treatment to the donor mice prior to
subcapsular transplant (7dsPT, Figure 2). No further M-T7, ASO
Ndst1
or control treatments
were given to the recipient BALB/c mice after engrafting.
Pathogens 2022,11, 588 15 of 20
Table 1. Renal allograft models; treatments and allograft numbers.
Renal Allograft Pretreatment-PTS and 7dsPT
Transplant
Donor Mouse–Recipient
Mouse
Treatment
Days
Follow
Up
Number of
Transplant
Procedures
PTS
Subcapsular transplant Pre-treatment Soak-1 h
C57BL/6-BALB/c Saline
3 days
4
Ndst1/C57Bl/6-BALB/c Saline 4
C57BL/6-BALB/c M-T7 4
C57BL/6-BALB/c ASONdst1 4
C57BL/6-BALB/c ASOScr 4
PTS
Subcapsular transplant Pre-treatment Soak-1 h
C57BL/6-BALB/c Saline
15 days
10
Ndst1/C57Bl/6-BALB/c Saline 4
C57BL/6-BALB/c M-T7 5
C57BL/6-BALB/c ASONdst1 7
C57BL/6-BALB/c ASOScr 4
7dsPT Subcapsular transplant-Pre-treatment
donor-7 days
C57BL/6-BALB/c Saline
3 days
6
C57BL/6-BALB/c M-T7 6
C57BL/6-BALB/c ASONdst1 6
C57BL/6-BALB/c ASOScr 6
7dsPT Subcapsular transplant-7days
Pre-treatment donor-7 days
C57BL/6-BALB/c Saline
15 days
6
C57BL/6-BALB/c M-T7 6
C57BL/6-BALB/c ASONdst1 6
C57BL/6-BALB/c ASOScr 6
PTS
Subcutaneous transplant Pre-treatment
Soak-1 h
C57BL/6-BALB/c Saline
3 days
6
Ndst1/C57Bl/6-BALB/c Saline 6
C57BL/6-BALB/c M-T7 3
C57BL/6-BALB/c ASONdst1 6
C57BL/6-BALB/c ASOScr 6
BALB/c-BALB/c Saline 5
PTS
Subcutaneous transplant Pre-treatment
Soak-1 h
C57BL/6-BALB/c Saline
15 days
2
Ndst1/C57Bl/6-BALB/c Saline 6
C57BL/6-BALB/c M-T7 4
C57BL/6-BALB/c ASONdst1 8
C57BL/6-BALB/c ASOScr 4
BALB/c-BALB/c Saline 6
PTS—pre-treatment of donor organ with flush and soaking X 1 h, 7dsPT-7 days pre-treatment of donor mouse,
Ndst1
/
—Ndst1 conditional knockout mouse, ASO
Ndst1
—antisense to Ndst1; ASO
Scr
—antisense control with
scrambled sequence.
4.2. Subcapsular Renal Allograft Transplant
Mice used included C57BL6/J, BALB/c and N-deacetylase-N-sulfotransferase-1 (Ndst1f/f
TekCre
+
or Ndst1
/
) at 8–12 weeks of age (Table 1; N = 50 subcapsular PTS renal trans-
plants, 20 with 3 days follow-up and 28 with 15 days follow-up; N = 48 subcapsular 7dsPT
renal transplants, 24 with 3 days follow-up and 24 with 15 days follow-up). Kidneys
were isolated after euthanasia from C57Bl/6 mice or Ndst1
/
, conditional knock-out
mice lacking Ndst1 expression in endothelial cells and myeloid precursors). Sections of
C57BL/6 mice kidneys were used as donors for BALB/c recipient mice; One C57BL/6
Pathogens 2022,11, 588 16 of 20
kidney divided into sections for implant into six recipient BALB/c mice [
35
]. Sections
were cut to incorporate all layers, cortex to hilum. Anesthetized BALB/c mice (Ketamine
(100 mg/mL, 120 mg/kg)/xylazine (20 mg/mL, 6 mg/kg) mixture) were shaved to create
a 2-inch area on the right side of the mouse over the kidney. The surgical site was cleaned
with Chlorhexidine Gluconate 2% solution and 70% ethanol and the surgeries performed in
a sterile field. A vertical incision approximately 0.7–1.0 cm in length midway between the
bottom of the rib cage and the iliac crest, the kidney exteriorized and a small incision made
in the renal capsule. A section of C57BL/6 mouse kidney, including medulla to outer cortex
kidney tissue, was then inserted into the subcapsular space and the kidney returned to the
abdomen and the surgical site closed with sterile sutures. Mice were given Buprenorphine
0.1 mg per/kg per mouse for pain control and subcutaneous sterile saline (200–500
µ
L)
to aid in recovery. Mice were kept on a heating pad until fully awake and then checked
daily for signs of pain and discomfort (hunching, piloerection, loss of appetite, weight loss,
etc.). If signs of excess pain were seen, repeat doses of buprenorphine were administered.
Mice were monitored by experienced veterinary staff as well as the experimental group.
There were two mice lost during transplant, one during subcutaneous implant and one
immediately before subcapsular transplant (2% mortality); both were deemed secondary to
anesthesia and surgery and were unrelated to specific treatments [
35
]. No mice were lost
after transplant.
4.3. Subcutaneous Renal Allograft Transplant
For subcutaneous transplant surgeries, the graft was implanted in a 0.25 inch pocket
incision in 1
×
1 inch shaved sections prepared on the back of the mouse between the
shoulders [
36
]. Half of one donor kidney was inserted into the subscapular pocket using
sterile forceps and the skin closed with two–three sutures. One mouse provided donor
kidney sections for four transplants (Table 1; N = 62 subcutaneous PTS renal transplants,
32 with 3 days follow-up, and 30 with 15 days follow-up). For the subcutaneous transplants
only soaking PTS treatments were used. Mice were checked daily for signs of discomfort
such as hunching, piloerection and weight loss. After 3 or 15 days post-transplant, mice
were euthanized using CO2 gas followed by cervical dislocation.
4.4. M-T7 Expression and Purification
M-T7 was expressed in CHO cells
in vitro
. The Myxoma virus-derived gene for the
37 kDa secreted glycoprotein M-T7 gene was inserted and M-T7 expressed in CHO cells.
Secreted M-T7 protein was isolated from concentrated, diafiltrated media (0.22
µ
M filter)
and loaded on a QFF 40 mL column using a peristaltic pump with UV monitoring during
FPLC. After loading, the column was washed with 25 mM Tris-HCl (pH7.4), 25 mM NaCl
buffer followed by 25 mM Tris-HCl (pH 7.4), 1 M NaCl (with graded washes from 5% up to
100%). Contaminants were removed by thiophilic binding mode using 3.6 M (NH
4
)
2
SO
4
,
20 mM Hepes, pH7.0 added slowly to QFF40 25%B eluate to a final concentration of
(NH
4
)
2
SO
4
of 0.7 M. After filtration through a 0.22
µ
M filter, the eluate was loaded on a
thiophilic column (8 mL), washed with 0.7 M (NH
4
)
2
SO
4
, 20 mM Hepes and M-T7 eluted
by 20 mM Hepes, pH 7.0. M-T7 protein was dialyzed and further purified on a ceramic
hydroxyapatite (CHT) column followed by dialysis against phosphate-buffered saline
(PBS). M-T7 isolates were 96% pure with a single band and dimers detected on SDS PAGE
(Figure 9). Endotoxin was assessed and below the limit of detection (0.125 EU/mL or
0.231 EU/mg). Final protein preps were filter sterilized using a 0.22 micron filter and stored
at 4 C for use within 1 week or stored at 80 C for long-term storage [79,37,38].
Pathogens 2022,11, 588 17 of 20
Figure 9.
M-T7—Gel electrophoresis illustrating M-T7 purification. Monomeric M-T7 bands are
present at approximately 35 kD with evidence for dimers at 70 kD.
4.5. Antisense to N Deacetylase Sulfotransferase-1 (Ndst1) ASONdst1 Construct Design
An ASONdst1 construct was developed and chemically modified to improve potency
and stability (developed by SY; Target Sequence NDST1 ASO: CTGCAACTTACTTTTA;
control ASO: GGCCAATACGCCGTCA). The ASO
Ndst1
was designed with molecular
composition and quality analysis for maximum stability, activity and lack of toxicity.
These modifications included a phosphorothioate (PS) backbone and ribose modification,
replacing cytosine with 5-methylcytosine (Figure 10A).
Figure 10.
ASO
Ndst1
sequence and activity. (
A
). Sequences for ASO
Ndst1
and ASO
Scr
Control;
(
B
). Significantly reduced Ndst1 expression is detected in normal, non-transplanted C57BL/6J mice
treated with ASO
Ndst1
demonstrated reduced Ndst1 expression on Taqman RT-qPCR. A greater
percentage reduction in Ndst1 expression is seen after ASO
Ndst1
treatment at doses above 30 mg;
(
C
). Doses at 10–50 mg are entirely safe in normal mice without transplant. AST and bilirubin were
increased at doses of 100 mg. Two-Way ANOVA w/Benjamini post-hoc vs. 0 mg. * p< 0.05,
** p< 0.01,
*** p< 0.001, **** p< 0.0001.
These ASOs have a 3-10-3 design, which means the first three and last three sugar
molecules were cET bridged nucleic acids and the middle 10 sugars were unmodified
deoxyribose nucleotides. NDST1 ASOs were synthesized and screened in cell culture for
in vitro
activity. This lead ASO was the most actively tolerated ASO in mice. The control
ASO was selected with the same chemistry but did not target any mouse gene. ASO
Ndst1
significantly reduced the detected Ndst1 expression in normal mice. (Figure 10B). ASO
Ndst1
Pathogens 2022,11, 588 18 of 20
was assessed for any adverse effects in normal mice, no significant adverse effects were
detected (Figure 10C).
4.6. Histopathology and Immunohistochemistry
Grafts were assessed for histological analysis with routine hematoxylin and eosin
(H&E) staining as well as immunohistochemical (IHC) staining. Total inflammatory cell
infiltrate area and scar (fibrotic) areas were measured and normalized to the total renal
allograft implant area [
7
9
,
12
,
13
,
35
,
38
]. The diameter of the inflammation and scar were
similarly measured and normalized to total graft diameter. The numbers of histologically
normal glomeruli were also counted in renal allografts. A histopathology score was
developed based upon the classifications used in the Banff criteria with assigned scores
ranging from 1+ up to a maximum of 4+ for detectable tubule or glomerular inflammation
or scarring (fibrosis), providing a score for the histopathology associated with acute damage,
inflammation and rejection.
IHC was performed for the detection of cell and rejection markers: F4/80 for macrophage;
CD3 for nonspecific T lymphocytes; Ly6G for neutrophils; CD19 for B lymphocytes and
C4d as a marker for transplant rejection. For IHC, the primary antibodies included the
following: Anti-mouse C4d Cat: HP8033; Ra pAb to F4/80 ab100790; Rb pAb to CD3m ab
5690 and secondary antibody: Goat antiRat IgG2a Hrp conjugated Cat: A110-109P. Sections
were examined using an Olympus BX51 microscope with 4
×
–100
×
objectives, a Prior
ProScan II stage and Olympus DP74 CMOS camera and cellSens software analysis system.
Staining for C4d was used as a secondary marker of rejection.
4.7. Statistical Analysis
Immunohistochemical analyses were read initially by MB, JY and AL and then read by
an investigator (IRZ) blinded to treatment and mouse strain. Significance in each parameter
was assessed by StatView version 5.0.1 (SAS Institute, Inc., Cary, NC, USA) using one-way
analysis of variance (ANOVA) with Fischer’s LSD (Least Significant Difference) comparison,
or a Student’s unpaired t-test. p< 0.05 was considered significant.
Author Contributions:
Conceptualization, E.L., S.A. and A.L.; Formal analysis, J.R.Y., W.C., M.B. and
A.L.; Investigation, I.R.Z., M.B., J.R.Y., J.K., K.M.L., W.C. and L.Z.; Resources, S.T.Y., K.M.L., J.D.-V.
and R.B.; Supervision, A.L.; Writing–original draft, I.R.Z., L.Z. and D.F.; Writing–review and editing,
I.R.Z., M.B. and A.L.; Methodology, H.C., J.R.Y., L.Z., D.W., J.K. and M.B.; Visualization, W.C. and
M.B. All authors have read and agreed to the published version of the manuscript.
Funding:
This work was supported by The American Heart Association (AHA) and The Enduring
Hearts Foundation through grant numbers 0855421E, 12GRNT120/0313, and 17GRNT33460327, as
well as startup funding from the Biodesign Institute, Arizona State University.
Institutional Review Board Statement:
All renal allograft procedures were approved by the Arizona
State University (ASU) Institutional Animal Care and Use Committee (IACUC) and conformed to
national, international and university guidelines for animal care. ASU protocol Number:20-1761R.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
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A coat of pericellular hyaluronan surrounds mature dendritic cells (DC) and contributes to cell-cell interactions. We asked whether 4-methylumbelliferone (4MU), an oral inhibitor of HA synthesis, could inhibit antigen presentation. We find that 4MU treatment reduces pericellular hyaluronan, destabilizes interactions between DC and T-cells, and prevents T-cell proliferation in vitro and in vivo. These effects were observed only when 4MU was added prior to initial antigen presentation but not later, consistent with 4MU-mediated inhibition of de novo antigenic responses. Building on these findings, we find that 4MU delays rejection of allogeneic pancreatic islet transplant and allogeneic cardiac transplants in mice and suppresses allogeneic T-cell activation in human mixed lymphocyte reactions. We conclude that 4MU, an approved drug, may have benefit as an adjunctive agent to delay transplantation rejection.
Chapter
Solid tissue transplant is a growing medical need that is further complicated by a limited donor organ supply. Acute and chronic rejection occurs in nearly all transplants and reduces long-term graft survival, thus increasing the need for repeat transplantation. Viruses have evolved highly adapted responses designed to evade the host’s immune defenses. Immunomodulatory proteins derived from viruses represent a novel class of potential therapeutics that are under investigation as biologics to attenuate immune-mediated rejection and damage. These immune-modulating proteins have the potential to reduce the need for traditional posttransplant immune suppressants and improve graft survival. The myxoma virus-derived protein M-T7 is a promising biologic that targets chemokine and glycosaminoglycan pathways central to kidney transplant rejection. Orthotopic transplantations in mice are prohibitively difficult and costly and require a highly trained microsurgeon to successfully perform the procedure. Here we describe a kidney-to-kidney subcapsular transplant model as a practical and simple method for studying transplant rejection, a model that requires fewer mice. One kidney can be used as a donor for transplants into six or more recipient mice. Using this model there is lower morbidity, pain, and mortality for the mice. Subcapsular kidney transplantation provides a first step approach to testing virus-derived proteins as new potential immune-modulating therapeutics to reduce transplant rejection and inflammation.
Article
Background Both the complement and the coagulation systems play important roles in the development of hyperacute or acute antibody‐mediated xenograft rejection. Atrase B is a novel metalloproteinase isolated from the venom of Naja atra . In this study, we investigated the inhibitory effects of atrase B on complement activation and coagulation, as well as the effect on xenograft survival in a discordant xenotransplantation model. Methods The in vitro anti‐complement activity of atrase B was evaluated using a normal human serum (NHS)‐mediated complement‐dependent cytotoxicity model with an immortalized porcine aortic endothelial cell line (iPEC) as the target. The in vivo inhibitory effects on complement activity and coagulation function were measured in rats after the administration of atrase B. Guinea pig hearts were transplanted heterotopically into Wistar rats with or without atrase B pre‐treatment. Results Pre‐treatment of the NHS with atrase B inhibited the cell lysis of iPECs in a dose‐dependent manner. FACS analysis showed that atrase B potently suppressed the deposition of C5b‐9, but not C3c and C4c, on iPECs. In vivo, atrase B‐treated rats showed a significant reduction in serum complement activity; markedly prolonged PT, APTT, and TT; and a decreased plasma level of fibrinogen. When compared to PBS treatment evaluated at study endpoint, atrase B treatment significantly delayed xenograft rejection and attenuated pathologic damage, the formation of platelet microthrombi, and the deposition of fibrin and C5b‐9. Conclusions The dual activities of anti‐complement and anti‐coagulation make atrase B a potential adjuvant therapeutic drug for use in xenotransplantation.