Single-armed salamo-like dioxime and its multinuclear Cu (II), Zn (II) and Cd (II) complexes: Syntheses, structural characterizations, Hirshfeld analyses and fluorescence properties

Article abstract of DOI:10.1002/aoc.5240

Three multinuclear Cu (II), Zn (II) and Cd (II) complexes, [Cu₂(L)(μ-OAc)]·CHCl₂ (1), [Zn₂(L)(μ-OAc)(H₂O)]·3CHCl₃ (2) and [{Cd₂(L)(OAc)(CH₃CH₂OH)}₂]·2CH

Full text of DOI:10.1002/aoc.5240

Received: 25 January 2019ꢀ ꢀ Revised: 17 March 2019ꢀ ꢀ Accepted: 24 March 2019
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DOI: 10.1111/ajt.15376
O R I G I N A L A R T I C L E
A clinically relevant murine model unmasks a
“two‐hit” mechanism for reactivation and dissemination of
cytomegalovirus after kidney transplant
Zheng Zhang1,2ꢀ| Longhui Qiu1ꢀ| Shixian Yan1ꢀ| Jiao‐Jing Wang1ꢀ| Paul M. Thomas3ꢀ|
Manoj Kandpal1,4ꢀ| Lihui Zhao1,4ꢀ| Andre Iovane1ꢀ| Xue‐feng Liu1,2ꢀ| Edward B. Thorp5ꢀ|
Qing Chen5ꢀ| Mary Hummel1,2,6ꢀ| Yashpal S. Kanwar5,7ꢀ| Michael M. Abecassis1,2,6
1Comprehensive Transplant Center, Northwestern University, Feinberg School of Medicine, Chicago, Illinois
2Department of Surgery, Northwestern University, Feinberg School of Medicine, Chicago, Illinois
3Department of Chemistry and Chemistry of Life Processes Institute, Northwestern University, Evanston, Illinois
4Preventive Medicine, Northwestern University, Feinberg School of Medicine, Chicago, Illinois
5Department of Pathology, Northwestern University, Feinberg School of Medicine, Chicago, Illinois
6Department of Microbiology and Immunology, Northwestern University, Feinberg School of Medicine, Chicago, Illinois
7Department of Nephrology, Northwestern University, Feinberg School of Medicine, Chicago, Illinois
Correspondence
Michael M. Abecassis
Reactivation of latent cytomegalovirus remains an important complication after
Email: Michael.Abecassis@nm.org
transplant. Although immunosuppression (IS) has been implicated as a primary cause,
we have previously shown that the implantation response of a kidney allograft can
lead to early transcriptional activation of latent murine cytomegalovirus (MCMV)
genes in an immune‐competent host and to MCMV reactivation and dissemination to
other organs in a genetically immune‐deficient recipient. We now describe a model
that allows us to separately analyze the impact of the implantation effect vs that of a
clinically relevant IS regimen. Treatment with IS of latently infected mice alone does
not induce viral reactivation, but transplant of latently infected allogeneic kidneys
combined with IS facilitates MCMV reactivation in the graft and dissemination to
other organs. The IS regimen effectively dampens allo‐immune inflammatory path‐
ways and depletes recipient anti‐MCMV but does not affect ischemia–reperfusion
injury pathways. MCMV reactivation similar to that seen in allogeneic transplants
combined with also occurs after syngeneic transplants. Thus, our data strongly sug‐
gest that while ischemia‐reperfusion injury of the implanted graft is sufficient and
necessary to initiate transcriptional reactivation of latent MCMV (“first hit”), IS is
permissive to the first hit and facilitates dissemination to other organs (“second hit”).
Funding information
Flow cytometry and histological analyses
were provided by the Northwestern
University Flow Cytometry Core Facility
and Mouse Histology and Phenotyping
Laboratory, respectively, both of which
are supported by National Cancer
Institute P30‐CA060553 awarded to the
Robert H Lurie Comprehensive Cancer
Center. This work is supported by NIH/
National Institute of Allergy and Infectious
Diseases P01AI112522 (Dr Abecassis) and
R01AI112911 (Dr v).
Abbreviations: IL, interleukin; HCMV, human cytomegalovirus; PCR, polymerase chain reaction; LC‐MS/MS, liquid chromatography–tandem mass spectrometry; ALS, anti‐mouse
lymphocyte serum; D+, donor infected with MCMV; DC , dendritic cell; DEX, dexamethasone; H3.3K36Me2, dimethylated H3.3K36; H3K79Me2, dimethylated H3.3K36; H3K9ac,
acetylated histone 3 lysine 9; H3K9Me2, dimethylated H3K9; IRI, ischemia–reperfusion injury; IS, immunosuppression; MCMV, mouse cytomegalovirus; POD, postoperative day; R−,
naive recipient; SG, salivary gland; TF, transcription factor.
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Am J Transplant. 2019;19:2421–2433.
amjtransplant.com
© 2019 The American Society of Transplantation ꢀ ꢀ
and the American Society of Transplant Surgeons

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K E Y W O R D S
animal models: murine, basic (laboratory) research/science, immunosuppression/immune
modulation, immunosuppressive regimens, infection and infectious agents ‐ viral:
Cytomegalovirus (CMV), infectious disease, ischemia reperfusion injury (IRI), kidney
transplantation/nephrology, signaling/signaling pathways, translational research/science
1ꢀ|ꢀINTRODUCTION
2.1ꢀ|ꢀMice
BALB/c (CD45.1 and CD45.2, H‐2d) and B6 (H‐2b) mice from Jackson
Cytomegalovirus (CMV) is a ubiquitous beta‐herpesvirus that es‐
tablishes lifelong latency in the host.1 CMV remains clinically silent
after convalescence from the initial infection but can reactivate from
latency, causing infectious complications in transplant patients.2‐4
CMV infection continues to be associated with significant morbid‐
ity and mortality after transplant.5‐7 Although both prophylactic
and preemptive strategies are effective in preventing and treating
infections posttransplant, all current antiviral regimens treat, rather
than prevent, reactivation; despite this, CMV mismatch continues
to adversely affect patient and graft survival.8 Several factors have
been previously associated with the occurrence of CMV reactivation
and disease in transplant recipients. These factors include seropos‐
itive donors/seronegative recipients (no preexisting CMV‐specific T
cell immunity), the organ type, and the intensity of the IS regimen,
among others,9 but the individual contribution of each as causative
factors remains elusive.
Laboratories (Bar Harbor, ME) were housed in the animal facility
at Center for Comparative Medicine, Northwestern University. All
mice were used according to protocols approved by the Institutional
Animal Care and Use Committee of Northwestern University.
2.2ꢀ|ꢀVirus and establishment of latency
The m157 deletion mutant MCMV (Δm157) strain was originally
obtained from Professor Ulrich Koszinowski (Ludwig Maximilians‐
University, Munich, Germany) and used as previously described.13,15
The Δm157 strain was used for generating latently infected donor
mice to avoid potential effect of MCMV resistance seen in B6 mice
due to interactions between Ly49H and the m157 protein.15,16 To es‐
tablish latency, BALB/c or B6 mice were infected (intraperitoneally)
with 1 × 107 plaque‐forming units of virus and were used as trans‐
plant donors 4‐6 months after infection. Latency was confirmed by
serology (Figure S1) and DNA analysis.10,14
Due to the species‐specificity of CMV, we and others have pre‐
viously used the murine CMV (MCMV) model to study the mech‐
anisms responsible for CMV reactivation from latency. Based on
cumulative data from our laboratory using a vascularized murine
kidney transplant model, we determined that the inflammatory re‐
sponse that results from graft implantation is required for MCMV
to become transcriptionally active after transplant, but this tran‐
scriptional reactivation in the graft did not progress to viral repli‐
cation, dissemination, and infection of the immune‐competent
recipient.10‐13 In subsequent studies, we discovered that transplant
of latently infected grafts into genetically immune‐compromised re‐
cipients resulted in transcriptional reactivation followed by viral rep‐
lication and dissemination.14 However, this latter model did not allow
us to address clinical relevant questions regarding the distinct role
of the inflammatory response associated with organ transplant pro‐
cess and the effect of IS. More recently, we developed a model that
included treatment of immune‐competent recipients with a clinically
relevant immunosuppression (IS) regimen. This model now allows for
the assessment of the individual contributions of alloreactivity, IS,
and ischemia–reperfusion injury (IRI) to CMV reactivation and infec‐
tion after transplant.
2.3ꢀ|ꢀKidney transplant
Kidneys from latently infected (D+) BALB/c or D+ B6 donor mice
were transplanted into binephrectomized naive (R−) BALB/c or R−
B6 recipient mice, respectively. The technical details of the surgery
have previously been described.17‐19 Tissues and blood samples
were collected at several predesignated endpoints posttransplant
for histopathological, viral, and molecular analyses. In all cases, the
contralateral donor kidney (not transplanted) was used as a control
for the graft.
2.4ꢀ|ꢀImmunosuppression
Recipients (with IS) were treated with a clinically relevant IS regi‐
men consisting of (1) FK506 (Tacrolimus, Astellas Pharma US,
Inc.), 3 mg/kg daily subcutaneously from day 0 to postoperative
day (POD)7, then every other day until the endpoints, (2) rabbit
anti‐mouse lymphocyte serum (ALS, Mybiosource, San Diego, CA),
4 doses of 300 μL intraperitoneal, starting from 12 hours before
transplant, then once every other day; and (3) dexamethasone
(DEX), 1 mg/kg intraperitoneal daily from POD0 to POD7, then
once every other day until euthanasia. Other recipients were not
treated with IS (without IS). Additional groups of latently infected
mice were also with the same IS regimen but did not undergo
2ꢀ|ꢀMATERIALS AND METHODS
An expanded Methods section is available in the Online Supplemental
Method.

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transplant. Control vehicle (PBS) was used for control groups.
Mice were euthanized at weekly intervals until 28 days after either
transplant or initiation of IS or PBS.
2.9ꢀ|ꢀTranscriptome analysis
Tissues from treated and untreated recipients were recovered,
snap‐frozen in liquid nitrogen, and stored at −80°C for RNA analysis.
RNA was quantified, quality was assessed, and genome‐wide RNA
expression was analyzed using Affymetrix MG‐430 PM mouse mi‐
croarrays as previously described.27
2.5ꢀ|ꢀQuantitative viral DNA analysis and
Plaque assay
Frozen tissues were processed for both DNA analysis by quantita‐
tive polymerase chain reaction (PCR) and plaque assays. The viral
DNA copy number per 1 million cells in each sample was normalized
by dividing the average number of MCMV IE‐1 copies by the aver‐
age GAPDH copy number and multiplying by 2 × 106 as previously
described.14
2.10ꢀ|ꢀCell isolation and flow cytometry
Cells from spleens or kidneys were isolated as previously described.19,28
Cells were then stained with the MHC‐tetramer (kb‐m38, NIH Tetramer
Core Facility) and various fluorescently conjugated antibodies includ‐
ing CD45 (30‐F11); CD45.1 (A20), CD45.2 (104), CD11b (M1/70), Ly6G
(1A8), F4/80 (T45‐2342), CD11c (HL3), MHCII (M5/114.15.2), CD4
(GK1.5), CD8 (53‐6.7), CD44 (1M7), CD3 (17A2), B220 (Ly‐5), and Ly6C
(HK1.4). A fixable viability dye eFluor 506 (Cat. 65‐0866‐14, eBiosci‐
ence) was used to gate out dead cells. Data were acquired with a BD
LSRII flow cytometer using BD FACSDiva software (BD Bioscience).
Flow cytometry data were analyzed with FlowJo v10 software.
2.6ꢀ|ꢀHistone analysis
Half of a mouse kidney was dissociated and lysed in nuclear isola‐
tion buffer. Histones were precipitated, air dried, and resuspended
for derivatization and digestion according to Garcia et al.20 The
resulting peptides were used for liquid chromatography–tan‐
dem mass spectrometry (LC‐MS/MS) analysis. Monitored pep‐
tides were selected based on previous reports.21,22 Raw MS files
were imported and analyzed in Skyline software with Savitzky‐
Golay smoothing.23 Peptide peak areas from Skyline were used
to determine the relative abundance of each histone modifica‐
tion by calculating the peptide peak area for a peptide of inter‐
est and dividing by the sum of the peak areas for all peptides
with that sequence; for example: H3K9ac relative abundance = H
3K9acK14un ÷ (H3K9unK14un + H3K9acK14un + H3K9unK14ac +
H3K9acK14ac). The relative abundances were determined based on
the mean of 3 technical replicates for each sample.
2.11ꢀ|ꢀHistology
Kidney samples were bisected transversely and processed and
stained with periodic acid–Schiff as previously described19 and eval‐
uated blindly by experienced renal pathologists (Y.S.K. and Q.C.) for
the morphologic characteristics of CMV infection.
2.12ꢀ|ꢀStatistical analysis
All statistical analyses were performed using GraphPad PRISM 7 soft‐
ware unless otherwise indicated. The Wilcoxon rank‐sum test was used to
compare the DNA copy levels between the with‐IS vs without‐IS groups
and controls. A P value <.05 was considered statistically significant. For
genome‐wide RNA expression, Affymetrix Mouse HT_MG‐430_PM mi‐
croarrays were normalized using Robust Multichip Average,29 and signal
filters of log2 < 3.8 were used to exclude probe sets with low signal inten‐
sities to filter out probe sets in the noise range. Pairwise class comparisons
were carried out using a 1‐way ANOVA by the Method of Moments30 in
Partek Genomics Suite 6.6. A false discovery rate of <5% was used for all
class comparisons. Pathway mapping to biologically significant pathways
was done using Ingenuity pathway analysis. All pathways were adjusted by
using the Benjamini‐Hochberg correction.
2.7ꢀ|ꢀBottom‐up proteomic analysis
Kidney tissue homogenate was tip sonicated and centrifuged for
protein extraction. The protein extract (50 μg) was precipitated
and then trypsinized to yield peptides. After lyophilization, pep‐
tides were reconstituted with 5% acetonitrile in 0.1% formic acid
for LC‐MS/MS data acquisition and processing. Protein tandem MS
data was queried for protein identification and label‐free quantifica‐
tion against the SwissProt Mus musculus database using MaxQuant
v1.6.0.16.24,25 Search results were validated with peptide and pro‐
tein false discovery rates both at 0.01. Proteins that were identified
with >1 peptide were subjected to a further statistical analysis using
Perseus software v1.6.0.7.26
3ꢀ|ꢀRESULTS
2.8ꢀ|ꢀPlasma protein analysis
3.1ꢀ|ꢀAllogeneic transplant of latently infected
kidneys in recipients treated with IS results in
reactivation of latent MCMV in the graft and
dissemination of MCMV to other organs
Plasma (70 μL) from treated and untreated or untreated transplant
recipients was frozen in liquid nitrogen and analyzed for inflamma‐
tory proteins at Ampersand Biosciences (Saranac Like, NY) by using
Luminex system and the Rodent MAP 4.0 Multiplex assay as previ‐
ously described.27
To extend our previous findings that alloreactivity induces early IE ex‐
pression in an immune competent model10,27 as well as dissemination

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of MCMV at later time points in a genetically immune‐compromised
model,14 we developed a new model of vascularized kidney transplant
that included immune competent mice treated with a regimen of clini‐
cally relevant IS, consisting of ALS, FK506 (calcineurin inhibitor), and
DEX (steroid). Kidneys latently infected with MCMV from B6 donors
(D+) were transplanted into naïve allogeneic BALB/c recipients (R−)
treated with IS (w/S) or without IS (wo/S) (Figure 1A). We observed a
significant increase in viral DNA copy number in transplanted kidneys
compared with the contralateral kidneys (control) by POD28. This dif‐
ference was enhanced in the with‐IS recipients vs without‐IS controls
(Figure 1B). We also detected viral DNA in the lung and salivary gland
(SG) of the with‐IS group as early as 7 days posttransplant, whereas
DNA detection was more delayed and less robust in the without‐IS
group (Figure 1C). Plaque assays demonstrated the presence of infec‐
tious viral particles (Table S1). A similar pattern in MCMV DNA am‐
plification was observed when kidneys from latently infected BALB/c
mice were transplanted into B6 recipients (Figure S2B). Treatment with
the clinically relevant IS reduced cellular infiltration and tissue injury,
which are typically seen in untreated allografts, and promoted viral dis‐
semination (Figure S2C and D). These data, using a newly developed
clinically relevant model, confirm previous findings that allogeneic
transplant of grafts latently infected with MCMV,10,27 in combination
with IS, results in viral reactivation and dissemination in the recipient.
after allogeneic transplant.10,27 To examine the impact of the IS
regimen on putative molecular pathways that might trigger tran‐
scriptional reactivation of MCMV, microarray and high‐through‐
put transcriptome analyses were performed on POD2 kidney
allografts. The heatmap in Figure 4A displays differentially ex‐
pressed genes clustered based on 4 distinct expression patterns.
Genes in cluster I are primarily involved in metabolic and hor‐
monal regulation pathways including glycine degradation, growth
hormone signaling, and insulin‐like growth factor‐1 signaling, and
they were significantly downregulated in both without‐IS and
with‐IS groups, compared with controls. Cluster II and cluster III
include genes in signaling pathways associated with host adap‐
tive immune responses (eg, helper T cells 1 and 2, allograft rejec‐
tion, costimulation, and B cell development) and genes involved
in the innate immune response (Toll‐like receptor signaling) and
cytokine signaling pathways (interleukin [IL]‐6, IL‐10, IL‐17, etc.),
respectively. Not surprisingly, the IS regimen inhibited activation
of these signaling pathways associated with both host adaptive
immune responses (cluster II) and innate immune responses (clus‐
ter III) (Table S3 [microarray analysis Excel file]). In contrast, IS
failed to downregulate genes involved in DNA damage, cell cycle,
and apoptosis (cluster IV). As shown in Figure 4B, transplants
for the with‐IS regimen had no effect in inhibiting activation of
signaling pathways associated with oxidative stress and DNA
damage. More specifically, ataxia telangiectasia–mutated and
RAD3‐like protein (ATR), activating transcription factor (ATF) 1/4,
checkpoint kinase (CHEK)1/2, and heat shock protein family A4
(HSPA4) were among common DNA damage‐ and stress‐associ‐
ated genes upregulated in both without‐IS and with‐IS conditions
(Figure 4C). These data suggest that cellular (endothelial cells)
stress responses in the graft (ie, oxidative stress or DNA damage)
secondary to IRI, rather than host allo‐immune responses, may
be implicated in triggering transcriptional reactivation of MCMV
after transplant in this model.
3.2ꢀ|ꢀIS regimen decreases numbers of host immune
cells and graft‐infiltrating cells and leads to loss of
CMV‐specific immunity
To address why the IS regimen promoted reactivation and dissemina‐
tion of the viruses, we examined the impact of IS on the host immune
response after allogeneic transplant. Splenic and graft‐infiltrating cells
were analyzed to determine the impact of IS on recipient immune re‐
sponses. At POD2, IS significantly reduced the number of recipient T
cells, whereas the number of myeloid cells in spleens was increased
(Figure 2A). In contrast, IS significantly inhibited the accumulation
of infiltrating cells in the allografts (Figure 2B). Specifically, IS signifi‐
cantly reduced the numbers of graft‐infiltrating DCs, macrophages,
and monocytes (Figure 2C) despite increased numbers in spleens.
Reduction of both splenic T cells and graft‐infiltrating cells persisted
at POD7 (Figure S3A). Both CD4 and CD8 memory cells (CD44hi) were
significantly decreased in recipients with‐IS (Figure S3B). Importantly,
the frequency of MCMV m38 specific CD8 T cells was significantly in‐
creased in the without‐IS but not in the with‐IS group, compared with
naive B6 mice (Figure 3A and B). These data suggest that the IS regi‐
men significantly affected CMV‐specific immunity in transplant re‐
cipients, potentially facilitating infection and systemic dissemination.
3.4ꢀ|ꢀIS regimen does not inhibit cellular histone
modifications associated with IRI
Changes in histone modifications are associated with MCMV re‐
activation.31‐33 To examine whether transplant IRI induces similar
changes in the histone landscape, we performed a comprehensive
histone analysis on the allografts from at 3‐48 hours posttransplant
of MCMV latently infected kidneys. Figure 5 shows that compared
with controls (contralateral kidneys), a rapid increase in permissive
acetylated histone H3 lysine 9 (H3K9Ac) levels at 3 hours (P < .01)
that persisted at 24 hours and 48 hours (P < .01) posttransplant
was observed in both with‐IS and without‐IS mice. Variant histone
H3, specifically H3.3, showed similar increases in levels of dimeth‐
ylated H3.3K36 (H3.3K36Me2) posttransplant. In contrast, levels
of dimethylated H3K9 (H3K9Me2) and H3K79Me2 demonstrated
reductions at 3 hours and remained low at 24 hours and 48 hours
posttransplant in allograft recipients treated without IS or with IS
compared with controls.
3.3ꢀ|ꢀIS regimen inhibits activation of host
inflammatory signaling in allogeneic transplants
but not of other IRI‐associated stress pathways
Transcriptional reactivation of MCMV can be detected as early
as 48 hours posttransplant, which were previously observed

ZHANG et Al.
ꢀꢁ ꢀ ꢀ2425
|
A
BALB/c (R-)
B6 (D+)
Con
Graft
wo/IS
w/IS
MCMV
Infection
DNA copy
Tx
-1
0
1
2
3
4
5
6
7
14
28
-120
ALS i.p. q.a.d.
Establishing
Latency
TAC + Dex i.p
q.d.
TAC + Dex
i.p q.a.d
B
wo/IS
w/IS
wo/IS
w/IS
wo/IS
w/IS
p<0.05
wo/IS
w/IS
C
p<0.05
p=0.01
p=0.05
p=0.01
p=0.1
p=0.05
POD7
POD14
POD28
F I G U R E 1 ꢀ Immunosuppression (IS) treatment promoted mouse cytomegalovirus (MCMV) reactivation and systemic dissemination after
D+‐to‐R− allogeneic transplants (allografts). Viral DNA in kidneys, lungs, and salivary glands (SGs) were quantified by quantitative PCR at
postoperative days (POD)7‐28 after allogeneic vascularized transplantation of D+ kidneys from B6 mice into R− BALB/c recipients (n = 5‐6/
time/group). A, Schematic of experimental setup. B, Viral DNA copy numbers in kidneys at POD0 (donor contralateral controls) and the
kidney allografts treated with IS (w/IS) or without IS (wo/IS) at PODs 7, 14, and 28. C, Viral DNA copy numbers in recipient lungs (r‐lung) and
salivary gland (r‐sg) from transplant recipients at PODs 7, 14, and 28

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ZHANG et Al.
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A spleen
w/IS
wo/IS
wo/IS
w/IS
1
0
8
6
4
2
0
0
0
0
0
0
**
**
**
**
C
D
4
+
C
D
8
+
D
C
M
o
n
o
M
a
c
G
r a
n
u
CD45.2
w/IS
B kidney
wo/IS
5
0
wo/IS
w/IS
**
4
3
2
1
0
0
0
0
0
CD45.2 (recipient)
C
D
4
5
. 2
C
D 4 5 . 1
CD45.1 (donor)
wo/IS
C kidney
w/IS
wo/IS
w/IS
2
0
**
1
1
5
0
**
**
*
5
**
3
**
2
1
0
C
D
4
+
C
D
8
+
D
C
M
o
n
o
M
a
c
G r a n u
CD45.2
F I G U R E 2 ꢀ Effect of IS on immune phenotypes in spleens and kidney allograft. Kidneys from BALB/c (CD45.1) were transplanted
into B6 recipients (CD45.2) with immunosuppression (IS) (w/IS) or without IS (wo/IS) (n = 4‐6/group). Cells isolated from spleens and
renal grafts were analyzed by flow cytometry. Frequencies of recipients’ T cells (gated on CD45.2+CD3+ cells) and myeloid cells (gated
on CD45.2+CD11b+ cells), including dendritic cells (DCs) (CD11c+MHCII+ DCs), Mac (CD11b+F4/80+ macrophages), Mono (CD11b+Ly6C+
monocytes), and Granu (Ly6G+ granulocytes) are calculated based on total number of live cells and normalized by tissue weight (mg). Data
in bar graphs are expressed as mean value with SEM. (*P ≤ .05 or **P < .01, wo/IS vs w/IS). A, Representative dot‐plot demonstrating
percentages of recipients’ T cells and myeloid cells and absolute number of subtypes in spleens at POD2. B, Representative dot‐plot
demonstrating percentages of recipients (CD45.2+) vs donor cells (CD45.1+) and their absolute numbers in the kidney allografts at POD2.
C, Representative dot‐plot demonstrating percentages of recipients’ T cells and myeloid cells and absolute number of subtypes (above
mentioned) in the kidney allografts at POD2

ZHANG et Al.
ꢀꢁ ꢀ ꢀ2427
|
A
wo/IS
3.4%
w/IS
0.6%
Naïve B6
0.1%
H-2Kb:m38
B
**
F I G U R E 3 ꢀ Immunosuppression (IS) suppresses host antiviral T cell responses following transplant. Cells from D+/R− kidney allografts
with IS (w/IS) or without IS (wo/IS) (n = 4‐6/group) were isolated and analyzed at postoperative (POD)7 for the frequency of viral specific T
cells MHC‐mouse cytomegalovirus (MCMV) by using m38 tetramer (Kb‐m38) staining. A, Representative dot‐plot showing the percentage of
MCMV‐specific CD8 T cells (gated on CD3+CD44+ cells). B, Absolute number in the transplanted kidneys wo/IS or w/IS; **P <.01. Naive B6
kidneys were controls
treated both with IS and without IS, whereas IS significantly inhib‐
3.5ꢀ|ꢀIS regimen does not inhibit cellular or plasma
ited expression of inflammatory mediators such as tumor necrosis
proteomic upregulation of pathways associated with
factor, IL‐5, IL‐6 and CXCL10 (Figure S4), which are associated with
MCMV reactivation
acute host immune responses, in mice treated with IS compared
To test the impact of the IS regimen on proteomic pathways pre‐
viously associated with MCMV reactivation,27 we performed 2
lines of proteomic analyses. Figure 6A shows a bottom‐up pro‐
teomic analysis of latently infected kidney allografts at 3 hours,
24 hours, and 48 hours. Oxidative phosphorylation and mitochon‐
drial dysfunction, typically activated pathways associated with
transplant IRI,34‐36 such as cellular stress responses, including the
DNA damage response, mitochondrial stress, and oxidative re‐
sponses, were all activated after transplant compared with con‐
trols in both mice treated with IS or without IS. Figure 6B shows
a protein analysis on plasma obtained from kidney recipients of
latently infected allografts 48 hours posttransplant. We observed
a significant increases in proteins related to endothelial cell ac‐
tivation and dysfunction, tissue damage (vascular endothelial
growth factor‐A, Serum Amyloid P component, Tissue Inhibitor
of Metalloproteinases 1, and Matrix metallopeptidase) in mice
with mice treated without IS.
3.6ꢀ|ꢀTreatment of latently infected mice with the
same IS regimen, in the absence of a transplant,
does not result in reactivation of MCMV
To determine whether the IS regimen itself was sufficient to induce
MCMV reactivation, BALB/c mice latently infected with MCMV
were treated for 28 days with the same IS regimen used for trans‐
plant recipients (Figure 7A). We did not detect significant differ‐
ences in viral DNA copy in kidney, liver, lung, spleen, and SG tissues
compared with latently infected mice treated with vehicle controls
(PBS) (Figure 7B). These data suggest that the IS regimen alone is
not sufficient to induce reactivation of latent MCMV and therefore
that the implantation response of a graft plays a role in reactivation
of latent MCMV.

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ZHANG et Al.
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A
Color key
con
wo/IS
w/IS
Clusters
I
low
high
-2
0
+2
Z-score
II
III
IV
w/IS wo/IS
B
Acute Phase Response
FXR/RXR Activation
DNA Double-Strand Break Repair
Chromosomal Replication
G2/M DNA Damage Checkpoint Regulation
CHK Proteins in Cell Cycle
F I G U R E 4 ꢀ Transcriptome profiling
on kidney allografts. Kidney tissues or
plasma samples were collected from
D+‐to‐R− kidney allografts treated with
immunosuppression (IS) (w/IS) or without
IS (wo/IS) at the indicated endpoints. The
contralateral latent donor kidney (con) was
recovered at the time of the transplant
(postoperative [POD]0). Pathways
NO & Reactive Oxygen Species
GADD45 Signaling
0
1
2
3
4
5
6
7
8
9
10
-log(B-H p-value)
C
with –log P value >1.3 (dashed line) are
statistically significant. A, Heat‐map of
deferentially expressed genes (DGF) in the
kidney allografts (n > 4/group) at 48 hours
posttransplant and clustered based on the
expression levels. B, Ingenuity pathway
analysis of selected genes in cluster IV
(heat‐map). C, Volcano plot showing
changes of genes associated with DNA
damage pathways in the transplants either
wo/IS (left) and w/IS (right)
wo/IS
w/IS
Log10(Fold changes)
vs without‐IS controls at 28 days posttransplant (Figure 8B and C).
Consistent with a role for IRI in MCMV reactivation, transcriptome
analysis at POD2 uncovered significant activation of DNA damage
and cellular stress pathways, which were similarly induced in synge‐
neic vs allogeneic transplants (Figure 8D, Supplemental XLS2). These
data suggest that IRI inherent to vascularized transplant is sufficient
to induce MCMV reactivation and that alloreactivity is not required.
3.7ꢀ|ꢀSyngeneic transplant of latently infected
kidneys in recipients treated with the IS regimen
results in reactivation of latent MCMV in the
graft and dissemination to other organs
To determine whether alloreactivity is required for MCMV reacti‐
vation and dissemination, we transplanted kidneys latently infected
with MCMV from BALB/c mice into syngeneic recipients with IS
and without IS by using a regimen equivalent to that used for allo‐
geneic transplants. Although viral DNA levels were generally lower
in transplanted kidneys and salivary glands compared with alloge‐
neic transplants, these were not statistically significant. Importantly,
however, we observed a significant increase in viral DNA copy num‐
ber in transplanted kidneys compared with the contralateral kidneys
(control) (Figure 8A). Viral reactivation was histologically confirmed
with the observation of intranuclear inclusion bodies in the synge‐
neic kidney graft with IS. Moreover, significant enhancement in viral
DNA amplification was observed in the lung and SG of the with‐IS
4ꢀ|ꢀDISCUSSION
CMV disease remains an important complication after transplant,
and IS has been and continues to be implicated as the primary factor
responsible for CMV reactivation and infection in this setting.37‐39
We have previously demonstrated that the inflammatory response
caused by the implantation of a kidney allograft can lead to early
changes in the transcriptional reactivation of immediate early viral
genes in an immune‐competent recipient27,40,41 and to viral infection

ZHANG et Al.
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wo/IS
w/IS
*
3.5
3
2.5
2
1.5
1
35
30
25
20
15
10
**
**
**
**
**
**
*
*
0
3
24
48
0
3
24
48
Time post transplant (hour)
Time post transplant (hour)
40
30
20
10
30
25
20
15
10
**
**
*
**
*
**
*
**
**
**
0
3
24
48
0
3
24
48
Time post transplant (hour)
Time post transplant (hour)
F I G U R E 5 ꢀ Quantification of cellular histone posttranslational modifications after transplant. Kidney tissues samples were collected
from D+‐to‐R− kidney allografts (n = 3/group/time) treated with immunosuppression (IS) (w/IS; red) or without IS (wo/IS; black) at 3, 24, and
48 hours posttransplant. Contralateral kidneys from the donor were controls (0 hours). Histones were extracted and analyzed with liquid
chromatography‐tandem mass spectrometry. The relative abundance of each histone modification is determined by calculating the peptide
peak area for a peptide of interest and dividing by the sum of the peak areas for all peptides with that sequence, based on the mean of 3
technical replicates with error bars representing the standard deviation. Data shown as percent of given modification in total pool of given
peptide that was quantified. *P < .05; **P < .01 compared with 0 hours controls
and dissemination in a genetically immune compromised recipient.14
We now describe our findings using a clinically relevant model that
allows us to separately analyze the individual mechanistic contribu‐
tions to CMV reactivation, infection, and dissemination of 3 inter‐
related components of a solid organ transplant: (1) clinically relevant
IS, (2) allo‐reactivity, and (3) IRI secondary to organ removal followed
by implantation.
inflammatory cytokines on transcription factor (TF) activation and
binding to the MIEP. More specifically, the MIEP for both HCMV and
MCMV, a critical check point in transcriptional control of the switch
between latency and reactivation, is a long and complex region that
encodes 2 differentially spliced genes with binding sites for several
activating TF, including nuclear factor‐κB, activating protein‐1, and
cyclic adenosine monophosphate response element binding pro‐
tein,47,48 that can be activated by tumor necrosis factor,45 lipopoly‐
saccharides,46 and allogeneic stimulation.10,27,49,50 Moreover, we
have previously reported that after murine kidney transplant plasma
proteomic data show evidence of a systemic inflammatory response
associated with CMV reactivation. In the current study, however,
supported by cellular transcriptome and plasma proteome analyses,
we found that although IS dampened pathways important in allo‐re‐
activity, it did not affect those associated with IRI. Moreover, using
a bottom‐up cellular proteomic approach, we observed significant
increases in proteins related to endothelial cell activation and tis‐
sue damage (vascular endothelial growth factor‐A, Serum Amyloid
P component, Tissue Inhibitor of Metalloproteinases 1, and Matrix
metallopeptidase) in recipients with IS and without IS. These obser‐
vations are particularly interesting given that endothelial cells are
known to be a site of MCMV latency in kidneys and may help explain
our findings after syngeneic transplants.
Our current data demonstrate that although treatment with IS
of latently infected mice alone does not induce viral reactivation,
transplant of latently infected allogeneic kidneys, which inherently
includes both IRI and alloreactivity, combined with a clinically rel‐
evant IS regimen leads to MCMV reactivation in the graft and viral
dissemination to other organs. The IS regimen effectively damp‐
ens allo‐immune inflammatory pathways and depletes recipient
anti‐MCMV but does not affect IRI pathways, including cellular his‐
tone modifications. MCMV reactivation similar to that seen in al‐
logeneic transplants combined with IS also occurs after syngeneic
transplants. Taken together, data derived from our newly developed
model strongly suggest, for the first time, that cellular stressors in‐
volving mitochondrial dysfunction and DNA damage mediated by
transplant‐related IRI is sufficient and required to induce reactiva‐
tion of latent MCMV in the graft, and viral dissemination to other
organs.
We and others have previously reported on the molecular mech‐
anisms by which both MCMV and human CMV (HCMV) establish
latency and reactivate from latency based on the regulation of their
major immediate‐early (IE) promoter (MIEP).10,42‐46 Transcriptional
reactivation of MCMV IE genes has been linked to the effect of
Recent studies have focused on epigenetic mechanisms related
to posttranslational histone modifications and chromatin remodel‐
ing.31‐33,48,51 The MIEP is heterochromatinized in latency and is as‐
sociated with marks of transcriptional repression whereby histone
protein cores compact and wrap viral DNA, limiting DNA accessibility

2430ꢀ ꢀ ꢀꢁ
ZHANG et Al.
|
A
B
F I G U R E 6 ꢀ Proteome profiling on kidney allografts and plasma protein analysis. Kidney tissues or plasma samples were collected from
D+‐to‐R− kidney allografts treated with immunosuppression (IS) (w/IS) or without IS (wo/IS) at the indicated endpoints. The contralateral
latent donor kidney (con) was recovered at the time of the transplant (postoperative [POD]0). Pathways with –log P value >1.3 (dashed line)
are statistically significant. A, Mass spectometry analysis for proteins extracted from the kidney tissues at 3, 24, and 48 hours posttransplant
(n = 3/group/time), followed by ingenuity pathway analysis. B, Multiplex proteomic analysis on plasma samples collected from D+/R− kidney
transplants at 48 hours posttransplant (n = 6‐8/group) and naive B6 (con). NS, not significant
for replication by repressing gene expression. H3 is hypoacetylated
at lysine 4, whereas H3 lysine 9 is hypermethylated during estab‐
lishment of latency of both MCMV and HCMV.32,33,52 We have pre‐
viously demonstrated that transplant of latently infected kidneys
caused a loss of the repressive H3K9me1 mark and an increase in the
activating mark H3K9Ac bound to both the MIEP and coding region
of the IE genes.31 Our current data show a rapid increase of permis‐
sive H3K9Ac marks at early time points, as well as in H3.3K36Me2,
a mark associated with DNA damage/repair typically found in ge‐
nomic regions exhibiting “active” or “poised” transcription,53 in the
A
cmv-
cmv+
∆m157
120days
wo/IS
w/IS
Day 28 DNA qPCR
F I G U R E 7 ꢀ Treatment with
immunosuppression (IS) regimen alone
failed to induced reactivation and
B
wo/IS
w/IS
dissemination of mouse cytomegalovirus
(MCMV) after transplant of latently
infected kidneys. BALB/c mice infected
with Δm157 mcmv virus were treated
with the same IS regimen (with IS [w/IS])
or PBS (without IS [wo/IS]) at 4 months
after the infection. Selected organs
were recovered at POD28. A, Schematic
of experimental setup. B, DNA copy
numbers in kidney, salivary gland (SG),
lung, liver, and spleen determined by real‐
time PCR

ZHANG et Al.
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|
F I G U R E 8 ꢀ Transplant ischemia–
reperfusion injury (IRI) is sufficient for
induction of mouse cytomegalovirus
(MCMV) reactivation and dissemination.
BALB/c mice infected with Δm157
mcmv virus were treated with the same
immunosuppression (IS) regimen (with
IS [w/IS]) or PBS (without IS [wo/IS])
at 6 months after the infection. DNA
copy numbers in kidney, salivary gland,
and lung were determined at day 28 by
real‐time PCR. A, DNA abundance in
postoperative day (POD)0 control kidney
(con) and POD28 after syngeneic D+/
R− kidney grafts treated w/IS or wo/
IS. B, Representative section (periodic
acid–Schiff staining, ×600 magnification)
of kidney graft w/IS showing an inclusion
body (arrow). C, DNA abundance
A
B
wo/IS
w/IS
P<0.01
Inclusion body
P=0.08
wo/IS
w/IS
P=0.01
C
at day 28 posttransplant recipients’
salivary gland (r‐sg) and lungs (r‐lung).
D, Transcriptome profiling and pathway
analyses were performed on untreated
D+/R− syngeneic kidney grafts (Syn) or
allogeneic (Allo) kidney grafts at POD2
(n = 3/group) as described in Figure 4.
Pathways with –log P value >1.3 (dashed
line) are statistically significant
D
Syn Allo
CHK proteins in cell cycle
DNA damage response
Chromosomal replication
Aryl Hydrocarbon Receptor
Cyclins & Cell Cycle Regulation
G2/M DNA Damage Checkpoint…
Mismatch Repair
0
1
2
3
4
5
6
7
8
9
10 11
-log(BH-corrected p-value)
presence or absence of IS. In contrast, levels of H3K9 Me2, a mark
of transcriptional repression, and H3K79m2, a mark associated with
transcription elongation and cell cycle,54 demonstrated significant
reductions at the same time points also in the presence or absence
of IS. Thus, our data, which are consistent with a previous report of
an acute kidney injury model,55 suggest that cellular histone modifi‐
cations known to be associated with IRI56‐58 and with transcriptional
reactivation of MCMV IE genes after transplant can occur indepen‐
dent of IS.
suggests that the enhancement of dissemination observed with IS
in both syngeneic and allogeneic transplants may be facilitated by
myeloid cells rather than by a T cell effect.
Although it is true that some MHC‐mismatched kidney trans‐
plants can survive long term without IS, we and others have re‐
ported that BALB/c recipients of B6 allografts develop more robust
acute rejection and that most recipients die as a result of rejection in
4 weeks, whereas most B6 recipients of BALB/c allografts typically
survive beyond 100 days with histological features of chronic rejec‐
tion. Despite different dynamics in graft rejection, these allografts
do develop histological evidence of acute rejection (lymphocyte
infiltration, tubulitis) during the early posttransplant period.18,19
Therefore, it is reasonable to hypothesize that allogeneic transplant
may accelerate or augment the process of MCMV reactivation as
opposed to syngeneic transplant, with or without IS, at the early
posttransplant time points. However, it is clear from our data that
syngeneic transplants, presumably through IRI mechanisms, are
sufficient to cause MCMV reactivation, and this is the focus of the
current study.
Our observations show that although IS alone does not result in
reactivation, it facilitates dissemination after a transplant. The clin‐
ically relevant IS regimen used significantly depleted both CD4 and
CD8 T cells while having little effect on myeloid or granulocytes and
significantly inhibited recruitment of host infiltrating cells in kidney
allografts. Further analysis showed that the frequency of MCMV
m38 specific T cells was increased in recipients of latently infected
allografts not treated with IS compared with the naive B6 kidney but
was diminished in kidneys from mice treated with IS. The finding that
IS depletes T cells, including memory T cells but not myeloid cells,

2432ꢀ ꢀ ꢀꢁ
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