Latent Herpesvirus Infection Augments Experimental
Kevin M. Vannella1, Tracy R. Luckhardt2, Carol A. Wilke2, Linda F. van Dyk3, Galen B. Toews2, and
Bethany B. Moore2,4
1Graduate Program in Immunology,2Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, and4Department of
Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan; and3Department of Microbiology, University of
Colorado School of Medicine, Aurora, Colorado
Rationale: No effective treatment exists for idiopathic pulmonary
fibrosis, and its pathogenesis remains unclear. Accumulating evi-
dence implicates herpesviruses as cofactors (either initiating or
exacerbating agents) of fibrotic lung disease, but a role for latent
herpesvirus infection has not been studied.
Objectives: To develop a murine model to determine whether latent
herpesvirus infection can augment fibrotic responses and to gain
insight into potential mechanisms of enhanced fibrogenesis.
before a fibrotic challenge with fluorescein isothiocyanate or bleo-
mycin so that the virus was latent at the time of fibrotic challenge.
Measurements were made after viral infection alone or after the
establishment of fibrosis.
Measurements and Main Results: gHerpesvirus is latent by 14 days
post infection, and infection 14 to 70 days before fibrotic chal-
lenge augmented fibrosis. Fibrotic augmentation was not de-
pendent on reactivation of the latent virus to a lytic state. Total
latently infected mice administered fibrotic challenge compared
with mock-infected mice that received fibrotic challenge. Latent
infection up-regulates expression of proinflammatory chemo-
kines, transforming growth factor-b1, and cysteinyl leukotrienes
in alveolar epithelial cells.
Conclusions: Latent gherpesvirus infection augments subsequent
fibrotic responsesinmice. Enhanced fibrosis isassociated with the
induction of profibrotic factors and the recruitment of fibrocytes.
the hypothesis that gherpesviruses can serve as initiating cofac-
tors in the pathogenesis of pulmonary fibrosis.
Keywords: chemokine; epithelial cells; fibrocyte; interstitial lung dis-
Idiopathic pulmonary fibrosis (IPF) is characterized by pro-
gressive and relentless lung scarring and is the most lethal
interstitial lung disease (1). Fibrotic lung disease likely results
from a dysregulated healing response to injurious events within
the lung, but initiating agents and molecular mechanisms
Herpesviruses have been suggested as cofactors for fibrosis
because their life cycle involves lytic replication followed by
establishment of lifelong latency with the potential for reac-
tivation. Previous studies have correlated the presence of
Epstein-Barr virus (EBV), cytomegalovirus, and human her-
pesviruses (HHV)-7 and -8 with IPF in human patients (2–5).
However, other studies find no association (6, 7). Mouse models
have been used to examine whether murine gherpesvirus-68
(gHV-68) contributes to the development of fibrotic lung
disease because gHV-68 is genetically and biologically similar
to EBV and HHV-8 (8). Although gHV-68 infection alone
does not cause fibrosis in wild-type mice, lytic gHV-68 pro-
motes the development of pulmonary fibrosis in the presence
of an exogenous injury insufficient to cause fibrosis on its own
(9). Furthermore, persistent lytic reactivation of gHV-68
induces fibrosis in Th2-biased mice (10). In addition, we have
recently shown that lytic gHV-68 infection can exacerbate
established pulmonary fibrosis (11). The mechanisms identi-
fied to contribute to lytic virus-induced exacerbation of
fibrosis have included Th2 bias, persistent reactivation, alter-
native activation of macrophages, and fibrocyte recruitment
Each of these findings provide clues about roles for lytic
gHV-68 infection in fibrosis, but most humans harbor latent
herpesviruses for their lifetime, and IPF normally occurs at
advanced age (14). A role for latent herpesviruses in fibrosis
has not been studied. Thus, we sought to establish an animal
model to test the hypothesis that latent gherpesvirus infection
can serve as a cofactor or an initiating agent of fibrosis in wild-
type mice. In these studies, we first infected mice intranasally
with gHV-68 and waited for latency to be established in the
lungs. We next challenged the mice with a fibrotic stimulus:
either fluorescein isothiocyanate (FITC) or bleomycin. Our
results demonstrate the ability of latent gHV-68 to augment
a subsequent fibrotic challenge and identify important latent
virus-induced alterations of lung epithelial cells that likely
promote fibrotic inflammation and extracellular matrix de-
AT A GLANCE COMMENTARY
Scientific Knowledge on the Subject
Herpesviruses have been associated with idiopathic pul-
monary fibrosis clinically, and animal models have sug-
gested a role for lytic infection as a fibrotic cofactor.
What This Study Adds to the Field
This study describes the first animal model for latent
herpesvirus-induced augmentation of lung fibrosis. Viral
augmentation of fibrosis is associated with fibrocyte re-
cruitment and induction of profibrotic mediators by la-
tently infected alveolar epithelial cells.
(Received in original form May 27, 2009; accepted in final form December 3, 2009)
Supported by NIH grants HL071586 (G.B.T. and B.B.M.), AI065543 (B.B.M.),
CA103632 (L.F.v.D.), and a research gift from the Martin Edward Galvin fund for
pulmonary fibrosis research. K.M.V. was supported by the Herman and Dorothy
Correspondence and requests for reprints should be addressed to Bethany B.
Moore, Ph.D., 4062 BSRB, 109 Zina Pitcher Place, Ann Arbor, MI 48109-2200.
This article has an online supplement, which is accessible from this issue’s table of
contents at www.atsjournals.org
Am J Respir Crit Care Med
Internet address: www.atsjournals.org
Vol 181. pp 465–477, 2010
Male mice (2–5 mo) were from Jackson Laboratories, Bar Harbor, ME.
Animal protocols were approved by the University of Michigan
Committee on the Use and Care of Animals.
Mice were anesthetized and 5 3 104plaque forming units (PFU) of
gHV-68 clone WUMS (American Type Culture Collection, Manassas,
VA), DORF72 (a v-cyclin mutant virus described previously (15)), or
v-cyclin marker rescue virus (‘‘MR’’) generated by recombination of
wild-type gHV-68 sequence, including the v-cyclin DNA, with a LacZ
cassette-containing virus, thus reconstituting an essentially wild-type
virus control (15), were suspended in 20 ml saline and delivered
intranasally to each mouse. Mock infection was 20 ml of saline.
Virus Plaque Assay
Lytic virus present in two lung lobes of infected mice was measured by
plaque assay (11).
Semiquantitative Real-Time Reverse
Transcriptase–Polymerase Chain Reaction
Semiquantitative real-time reverse transcriptase–polymerase chain re-
action (RT-PCR) was performed and the results quantified using the
DDCTmethod as described in Reference 16. The sequences for all
primers and probes are in Table 1. Samples were run in duplicate or
FITC and Bleomycin Injections
FITC and bleomycin inoculations were performed intratracheally as
described previously (17).
Total collagen measurements were made via hydroxyproline assay (17).
Lungs were enzymatically digested using collagenase and DNase (18).
Leukocytes from collagenase digests or bronchoalveolar lavage (BAL)
were incubated 15 minutes on ice with Fc block (1:100 dilution, clone
24G2; BD PharMingen, San Diego, CA) before surface staining with
CD45-PerCPCy5.5 (1:500 dilution, BD PharMingen) followed by
fixation/permeabilization with BD PharMingen Cytofix/cytoperm kit
according to manufacturer’s instructions. Cells were blocked with goat
IgG (1:2,000 dilution; Jackson ImmunoResearch, West Grove, PA)
before staining with rabbit anti-mouse collagen 1 (1:400 dilution;
(1:4,500 dilution; Jackson ImmunoResearch, isotype control). Cells
were analyzed on a FACScan (BD Biosciences, Mountain View, CA).
PA) or rabbitIgG
Hematoxylin and eosin and trichrome staining were performed as
described previously (18).
Chemokines CCL2, CCL12, and transforming growth factor (TGF)-b1
were measured by specific ELISA (DuoSet ELISA; R&D Systems
Minneapolis, MN). Cysteinyl leukotriene (CysLT) measurements were
made using EIA kits (Cayman Chemical, Ann Arbor, MI). Lower limit
of detection was 10 pg/ml. In some airway epithelial cell (AEC)
cultures, 5 mM Ca21ionophore (A23187) was added for 1 hour in
serum-free media as a maximal stimulus for LT synthesis.
Type II AECs were isolated as previously described (19, 20). Cells
cultured on fibronectin for 24 hours were greater than 88% positive for
e-cadherin, 3% positive for vimentin, and 0.5% positive for CD45.
Western blot analysis confirmed e-cadherin expression and showed no
a-smooth muscle actin.
Mink lung epithelial cells stably transfected with plasminogen activator
inhibitor (PAI)1 promoter-driven luciferase gene were mock infected
or infected with gHV-68 (multiplicity of infection of 0.005 or 0.05).
After 48 to 72 hours of infection, cell lysates were analyzed forTGF-b1
Complete media is Dulbecco’s modified Eagle medium (Lonza;
Walkersville, MD) with 10% fetal calf serum, 1% penicillin-strepto-
mycin, 1% L-glutamine, and 0.1% amphotericin B (Lonza). Serum-free
media is Dulbecco’s modified Eagle medium with 0.1% bovine serum
albumin (Sigma), 1% penicillin-streptomycin, 1% L-glutamine, and
0.1% amphotericin B.
Statistical significance was measured by analysis of variance (three or
more groups) or Student t test (two groups) using Graphpad Prism 3
software; data represent mean 6 SEM; P less than 0.05 was considered
gHV-68 Infection Is Latent in the Lung by 14 Days
To test the effect of latent gherpesvirus infection on sub-
sequent pulmonary fibrosis, we first confirmed when lytic virus
was cleared from the lungs of normal mice infected with gHV-
68. After intranasal inoculation with 5 3 104PFU, we were not
able to detect productive gHV-68 infection in the lung by
a standard plaque assay by 14 days post infection (d.p.i.)
(Figure 1A). Using real-time RT-PCR, we next measured viral
gene expression in the lung over the first 70 d.p.i. and
normalized these values to the expression level of each during
lytic replication at Day 3. Expression of the lytic gene DNApol
was substantially reduced by 14 d.p.i. (Figure 1B). Expression
of the M3 gene, which is expressed during both lytic and latent
infection, also diminished more slowly over time (Figure 1C).
Herpesvirus latency is defined by the expression of latency-
associated genes at higher levels than lytic-associated genes
when preformed lytic virus is not present. The normalization in
TABLE 1. PRIMERS AND PROBES FOR SEMIQUANTITATIVE
REAL-TIME REVERSE TRANSCRIPTASE–POLYMERASE
M3 F. primer
Definition of abbreviations: F. 5 forward; R. 5 reverse.
466 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINEVOL 1812010
fibrosis. We speculated that this might be the result of focal areas
of infection of lung AECs. Our results confirm that AECs are
a reservoir for latent gHV-68 infection in the lung and that latent
We isolated AECs at 14 and 21 d.p.i. The level of M3 gene
expression declines in AECs isolated during the third week after
infection when compared with AECs isolated 2 weeks post
infection. The decline could represent death of infected AECs
in vivo or possible differentiation to fibroblasts via epithelial to
mesenchymal transition (36). It could also be indicative of
a gradual silencing of viral gene expression at later time points
during latency. Our data agree with another study that demon-
strates AECs to be a reservoir of latent gHV-68 for at least 54
d.p.i. in vivo (26). EBV has previously been localized to AECs of
patients with IPF and has been associated with endoplasmic reti-
culum stress,the unfolded protein response, and a worseprognosis
(37, 38). Specifically, the expression of EBV latent membrane
protein 1 in AECs is associated with poor prognosis (39).
We hypothesized that latent gHV-68 alters the AEC phe-
notype to contribute to the augmentation of fibrocyte numbers,
inflammation, and TGF-b1 we observed in the whole lungs. Our
results demonstrate that latent infection of AECs induces
transcription of CCL2 and CCL12 mRNA, production of more
TGF-b1, and production of more cysLTs. Data collected by
Antoniades and colleagues and Mercer and colleagues corrob-
orate our findings by showing AECs are a prominent source of
CCL2 during pulmonary fibrosis (35, 40). We observed a delay
in the induction of CCL2 gene expression until 21 d.p.i. in
AECs; however, total CCL2 mRNA levels in the lungs of
latently infected mice were elevated by 15 d.p.i. Thus, much of
the CCL2 gene expression in the lung during this early period
after infection may be contributed by inflammatory cells rather
than AECs. We also found that AECs harvested from latently
infected mice produce significantly more total TGF-b1 than
AECs from mock-infected mice. In addition, we were able to
use mink lung epithelial cells stably transfected with a reporter
construct encoding a luciferase gene with a PAI1 promoter to
demonstrate that active TGF-b1 was being produced directly by
AECs after gHV-68 infection. We were unable to study latent
infection in vitro because the virus eventually destroyed these
cells via lytic replication during the second week of culture. Our
findings are consistent with the studies of Malizia and colleagues
who found that AEC injury with EBV up-regulates TGF-b1
expression in a human cell line (38). TGF-b1 is the most
powerful known promoter of extracellular matrix secretion and
a number of other fibrogenic processes (41, 42), and we expect
that gHV-68–induced TGF-b1 production is one mechanism that
contributes to the augmentation of fibrosis in our model.
We also demonstrated that AECs harvested from the lungs
of latently infected mice produce significantly more cysLTs than
AECs harvested from the lungs of mock-infected mice. This is
the first description of the ability of AECs to produce LTs.
CysLT levels are elevated in patients with IPF (27), and LT-
deficient mice are protected from experimental fibrosis (28, 29).
It is possible that the increased cysLT production in AECs
induces the secretion of TGF-b1. Leukotriene D4 has been
shown to induce TGF-b1 production in human bronchial
epithelial cells (43). The production of cysLTs by latently
infected AECs is likely to promote fibrosis via effects on
mesenchymal cells. We have previously reported that cysLTs
can cause fibrocytes to proliferate (29). Additionally, cysLTs are
known to induce fibroblast proliferation (44) and collagen
In sum, our model provides evidence that a latent gherpesvirus
infection can act as a cofactor in the development of sub-
sequent pulmonary fibrosis, and our results demonstrate for
the first time that short-term or long-term latency significantly
augments a subsequent fibrotic stimulus in immunocompetent
mice even if the stimulus is sub-threshold. Moreover, our
results are the first to identify potential mechanisms whereby
latent gherpesvirus infection can augment subsequent fibrotic
responses. We interpret our findings to suggest that the de-
velopment of fibrosis in this model requires at least two hits.
The first hit may involve the enhanced inflammatory response
and alteration of AECs to promote a profibrotic milieu. The
enhanced production of chemokines, TGF-b1, and cysLTs by
latently infected AECs may then drive fibrotic responses via
direct effects on mesenchymal cells. Figure 15 provides a hypo-
thetical model for how latently infected AECs may promote
fibrogenesis. These effects would be further amplified and/or
may become more persistent in the setting of a second hit with
a fibrotic or sub-threshold fibrotic stimulus.
Clinical data regarding an association of IPF with EBV in
particular are controversial. Because up to 95% of the popula-
tion harbors EBV in circulating B cells, the studies that are the
most informative look at lung tissue rather than blood. Several
studies have found evidence of EBV infection in the lungs of
patients with IPF at levels significantly higher than control
patients (2–5). These data suggest that EBV may be preferen-
tially maintained in the lungs of patients who develop IPF. A
different study suggested that presence of the EBV latent
membrane protein 1 correlated with more rapid progression
of IPF (39). However, two other studies found no evidence of
EBV in archived IPF tissue sections (6, 7). Thus, it is not clear
whether the discrepancies in the human literature represent
technical differences, geographical differences, disease hetero-
geneity, or perhaps smaller sample size in the negative studies.
Although gHV-68 infection is not identical to infection with
EBV or HHV-8, and FITC inoculation does not recapitulate all
aspects of IPF fibrogenesis, this model can be used to better
understand particular cell types or profibrotic factors critical to
virus-induced augmentation of fibrosis. The studies by Mora
and colleagues in Th2-biased mice suggested that antiviral
therapies may improve fibrotic outcomes by limiting viral
reactivation (13). In contrast, our studies suggest that viral
reactivation is not critical to the augmentation of fibrosis in
wild-type mice at least during early latency; thus, antiviral drugs
may not be as effective as previously hoped. A more fruitful
area for therapeutic investigation may be to try to find
mechanistic pathways that are similar in both lytic and latent
viral exacerbation of fibrosis and exploit these common path-
ways for therapeutic intervention.
Conflict of Interest Statement: K.M.V. does not have a financial relationship with
a commercial entity that has an interest in the subject of this manuscript. T.R.L.
does not have a financial relationship with a commercial entity that has an interest
in the subject of this manuscript. C.A.W. does not have a financial relationship
with a commercial entity that has an interest in the subject of this manuscript.
L.F.v.D. does not have a financial relationship with a commercial entity that has an
interest in the subject of this manuscript. G.B.T. does not have a financial
relationship with a commercial entity that has an interest in the subject of this
manuscript. B.B.M. has received consultancy fees from Centecor and Pfizer for up
to $1,000 and an industry-sponsored grant from Centecor ($100,001 or more).
Acknowledgment: The authors thank Dr. Thomas A. Moore for help with the
gating and analysis of flow cytometry samples.
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Vannella, Luckhardt, Wilke, et al.: Latent Virus Augments Fibrosis 477