Published online before print April 24, 2008
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,1
* Centre for Infectious Diseases,
Division of Veterinary Clinical Sciences, Royal (Dick) School of Veterinary Studies, and
The Institute of Immunology and Infection Research, University of Edinburgh, United Kingdom; and
Division of Medical Microbiology, School of Infection and Host Defence, University of Liverpool, United Kingdom
3Correspondence: Centre for Infectious Diseases, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Summerhall, Edinburgh, EH9 1QH, UK. E-mail: tony.nash{at}ed.ac.uk
ABSTRACT
Murine gammaherpesvirus 68 (MHV-68) is a natural pathogen of rodents closely related to the human 
herpesviruses Kaposi’s sarcoma-associated herpesvirus and EBV. Following intranasal infection, the virus replicates in the lung epithelium prior to establishing latent infection in lymphoid tissue. Infection of mice deficient in IFN-R signaling (IFN-R–/–) results in a multiple organ fibrosis, in which the spleen is severely affected. We show here that by Day 12 postinfection, prior to development of fibrosis in the spleens of IFN-R–/– mice, different subsets of splenic macrophages (M
s) are morphologically activated and enter latently infected germinal centers (GCs). Ms coexpressing arginase I (ARG1), a marker of alternative activation of Ms, and murine M markers F4/80, ER-TR9, and MOMA-1 are found in GCs of IFN-R–/– mice but not of wild-type mice. Quantitative RT-PCR of spleen RNA confirms induction of ARG1 and in addition, shows up-regulation of found in inflammatory zone 1/resistin-like molecule-
, tissue inhibitor of metalloproteinase-1, matrix metalloproteinase-12, fibronectin, and factor XIIIA in IFN-R–/– mice. In contrast, inducible NO synthase, associated with classical M activation, is up-regulated following infection of wild-type mice but not IFN-R–/– mice. Concomitant with the aaMs, transcription of the Th2 cytokines IL-13, IL-21, and IL-5 is up-regulated. Thus, in the absence of IFN-R signaling, MHV-68 initiates a Th2 immune response, leading to alternative activation of macrophages and induction of fibrosis. This system provides an important model for studying the pathogenesis of fibrosis initiated by a latent herpesvirus infection.
Key Words: Th2 • virus infection • arginase 1 • fibrogenesis • spleen
INTRODUCTION
Murid herpesvirus-4, commonly known as murine gammaherpesvirus-68 (MHV-68), is closely related to the human gammaherpesviruses Kaposi’s sarcoma-associated herpesvirus and EBV [1
, 2
]. MHV-68 infection of laboratory mice has been established as a small animal model to study the natural history of gammaherpesviruseses in their host. Following intranasal infection, the primary lesion observed in immunocompetent mice is interstitial pneumonia [3
], resolving by the 2nd week postinfection. Clearance of infection from the lung is mediated by CD8+ T cells [3
]. From the lung, the virus is carried to the lymphoid organs, where it establishes latent infection. Splenomegaly is observed during the 2nd week of infection following a rapid expansion of latently infected B cells. The splenomegaly is characterized by a large increase in B and T cell populations, and CD4+ T cells are essential for this pathological development [4
]. Latently infected B cells are mainly located in the germinal centers (GCs) of lymphoid organs, especially the spleen [5
6
7
8
]. Macrophages (Ms), dendritic cells (DCs), and lung epithelial cells are also sites of viral persistence [9
, 10
].
Previous work in our laboratory has shown that in mice lacking the IFN-R, MHV-68 infection results in a number of novel pathologies [11
12
13
]. Within the lung, in addition to the interstitial pneumonia observed in wild-type mice, interstitial and intra-alveloar fibrosis occurs with peak pathology detectable 2 weeks after infection following clearance of lytic virus. In the mediastinal lymph node and spleen, fibrosis occurs during the 3rd week of infection. This coincides with splenomegaly and the establishment of latent infection in wild-type mice and represents a critical phase in the virus life cycle, where there is an initial expansion of latently infected cells prior to establishment of long-term latency. In MHV-68-infected IFN-R–/– mice at this time in the infection, the mediastinal lymph node and spleen become severely depleted of cells, and collagen is deposited throughout these tissues. Coincidentally, the latent viral load in the spleen is 10–100 times that detected in wild-type mice during the 3rd week of infection. By 4 weeks postinfection, the viral load is controlled with only a few latent cells detected thereafter. Fibrotic changes are also detected in the liver with extensive portal fibrosis occurring 3–4 weeks postinfection. Of these pathologies, the most dramatic are those within the lymphoid system, the major site of latent virus infection. We have, therefore, focused on understanding the cellular and molecular events underlying fibrosis in the spleen. Depletion of CD4 and CD8 T cells prior to virus infection abrogates pathology, indicating a role for the T cell response in initiation of fibrosis. Analysis of cytokines and chemokine profiles in the spleen showed that levels of cytokines commonly associated with fibrosis, TGF-β1, TNF-, and IL-1β, were significantly elevated from the onset of fibrotic changes [13
]. However, the mechanism underlying this fibrosis is not understood. A particularly interesting, novel aspect of the fibrotic disease occurring in these mice is the resolution of fibrosis, which occurs in all tissues as the latent viral load rapidly declines following the intense fibrotic response around Day 20.
Other studies have also identified unique MHV-68-related pathologies in the absence of IFN- signaling. Intriguingly, these are related to the genetic background of the mice and the route of infection. The studies reported above relate to mice on the 129/Sv/Ev background. However, IFN-R–/– mice on the C57BL/6 background develop chronic pulmonary fibrosis following intranasal infection [14
], and i.p. infection of 129/Sv/Ev results in chronic arteritis of large blood vessels as well as lymphoid fibrosis in 60% of infected mice [15
]. These findings indicate an important relationship between gammaherpesvirus infection and fibrotic disease in hosts with a compromised immune response.
Fibrosis is an important tissue response affecting many vital organs, causing a significant level of morbidity and mortality in human beings. The etiology of many of these conditions is unknown [16
]. An association between fibrotic diseases and herpesviruses has been suggested, and in particular, strong evidence is available to link herpesviruses and idiopathic pulmonary fibrosis (IPF) in man [17
18
19
]. Here, we have studied splenic fibrosis induced in 129/Sv/Ev IFN-R–/– mice following intranasal MHV-68 infection to explore the cellular and molecular events associated with herpesvirus-induced fibrosis.
Activation of Ms and T lymphocytes has been shown to precede fibrotic changes in IPF in man [20
]. Alternatively activated Ms (aaMs) are of particular interest, as the presence of Ms with this phenotype is consistent with a role in tissue repair/fibrosis [21
, 22
]. Arginase 1 (ARG1) is a typical marker for aaMs [21
, 23
24
25
]. ARG1, like ARG2 and inducible NO synthase (iNOS), can metabolize L-arginine. In classically activated Ms, L-arginine is metabolized by iNOS to produce citrulline and NO, one of the principal cytotoxic mechanisms of these cells [26
, 27
]. In aaMs, L-arginine is metabolized by ARG1 into L-ornithine and urea. L-ornithine can be further metabolized to proline, which is an important amino acid required for collagen synthesis, and polyamines, which aid cellular proliferation. Indeed, Hesse et al. [28
] have shown in vitro proline production by aaMs under strict arginase control. aaMs are thus optimally suited to support tissue repair/fibrosis. In addition to ARG1, aaMs produce found in inflammatory zone 1 (FIZZ1)/resistin-like molecule- (RELM) [21
, 23
, 24
], a Th2-dependent protein that has also been strongly implicated in fibrosis [29
, 30
].
The present study was undertaken to investigate the involvement of Ms in MHV-68-induced fibrosis in the spleens of IFN-R–/– mice. Our data show different subsets of activated Ms migrating into latently infected GCs of IFN-R–/– mice. This event coincides with the detection of ARG1 and FIZZ1 expression in spleens from IFN-R–/– mice but not in spleens from wild-type, infected mice, indicating development of aaMs in spleens of IFN-R–/– mice undergoing fibrogenesis. Consistent with the induction of this M activation pathway, we show that IL-13, IL-5, and IL-21 as well as CCR4 expression are up-regulated in IFN-R–/– mice but not wild-type mice. Thus, MHV-68 infection in the absence of IFN-R signaling induces a Th2 response, which drives splenic aaMs, resulting in fibrotic disease in the spleen.
MATERIALS AND METHODS
Mice and virus
Wild-type mice and IFN-R–/– mice on the 129/Sv/Ev background were purchased from B and K Universal (Hull, UK) and bred in-house. Virus working stocks were prepared by infection of BHK-21 cells at a low multiplicity of infection (0.001 pfu/cell) with MHV-68 clone G2.4 [31
]. Infectivity of the virus was measured by plaque titration using BHK-21 cells grown in Eagle’s MEM containing 10% (v/v) tryptose phosphate broth and 10% (v/v) newborn calf serum at 37°C in 5% CO2 for 4 days.
Infection and sampling of mice
All experiments were carried out under a UK Home Office license in accordance with the Animals (Scientific Procedures) Act 1986. Age-matched IFN-R–/– and wild-type mice were anesthetized with halothane (Rhone Merieux Ltd., Harlow, UK) and inoculated intranasally with 4 x 105 pfu MHV-68 in 40 µl sterile PBS. For immunohistochemistry studies, the mice were killed by CO2 asphyxiation on Day 0 (naïve), 8, 10, 12, 14, 16, 18, or 20 p.i., and spleens were fixed in buffered formal saline and embedded in paraffin. Sections (5 µm) were used for immunohistochemistry studies. For mRNA analysis, another group of mice infected as above was killed after 0 (naïve), 8, 12, 16, 20, or 35 days (three mice per time-point), and spleens were collected in RNAlater (Ambion, UK) for RNA isolation and subsequent real-time RT-PCR.
Immunohistochemistry
Indirect immunoperoxidase techniques were used to demonstrate cells expressing F4/80, ER-TR9, MOMA-1, and ARG1 in spleen sections as described previously [32
33
34
35
36
]. Briefly, the paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated through a graded series of ethanol. Endogenous peroxidase was quenched by immersing the sections in 0.6% H2O2 (Sigma Chemical Co., Poole, Dorset, UK) in methanol for 30 min. Antigen unmasking was carried out using 0.1% protease IV (Sigma Chemical Co.) in TBS at pH 7.6 for 5 min. Primary antibodies against F4/80 (clone A3: 1) and MOMA-1 were obtained from Serotec (UK). Polyclonal antibody raised in rat against ER-TR9 was obtained from BMA Biomedicals (Switzerland). The antibody against ARG1 was a kind gift from Dr. Tomomi Gotoh of Kumamoto University (Japan). It was raised in a rabbit against recombinant human ARG1 and cross-reacts with rat and mouse ARG1 [37
]. The antibodies against F4/80, MOMA-1, and ER-TR9 were diluted in 2% normal rabbit serum in TBS at a concentration of 1:20 (v/v), 1:10 (v/v), and 1:1000 (v/v), respectively. The primary antibody against ARG1 was diluted at a ratio of 1:250 (v/v) in 2% normal goat serum in TBS. After incubation with the primary antibody for 2 h, the sections were washed in TBS, and secondary antibodies were applied for 30 min. F4/80, MOMA-1, and ER-TR9 were detected with biotinylated rabbit anti-rat IgG and ARG1 with biotinylated goat anti-rabbit IgG. The conjugated secondary antibodies were visualized using ABC reagent (Vector Laboratories, Peterborough, UK), as per the manufacturer’s instructions. Vector Nova Red (Vector Laboratories) was used as the substrate for demonstration of a positive reaction. Finally, the sections were counterstained with hematoxylin. For double-immunofluorescent staining of ARG1 plus F4/80, MOMA-1, or ER-TR9, the detection of ARG1 was carried out as described above with overnight incubation of primary antibody and detection with biotinylated secondary antibody and streptavidin-conjugated Alexa Fluor 488 (Invitrogen, Paisley, UK, 1:100 v/v). Detection of F4/80 was performed after ARG1 detection as described above except for final detection with streptavidin-conjugated Alexa Fluor 594 (Invitrogen, 1:100 v/v). Picrosirius Red staining was carried out by standard methods.
RNA isolation and real-time RT-PCR
RNA isolation from spleens was carried out using a Tissue RNeasy mini kit (Qiagen, Crawley, UK) according to the manufacturer’s instructions. RNA was resuspended in RNase-free water and treated with 10 U DNase 1 (DNA Free, Ambion) for 1 h at 37°C. The integrity of RNA was checked by the Agilent 2100 bioanalyzer. RNA (1 µg) was used for cDNA synthesis using Superscript III (Invitrogen) in a final volume of 20 µl. To verify genomic DNA decontamination after DNase treatment and check the quality of the cDNA, PCR for the β-actin gene was performed using the following primer set: forward: 5'-TGT GAT GGT GGG AAT GGG TCA-3' and reverse: 5'-TTT GAT GTC ACG CAC GAT TTC-3'. The PCR reaction was carried out in 50 µl Taq polymerase buffer containing 2 µl cDNA, 5 U Taq DNA polymerase (Invitrogen), 100 µM dNTP, 3 mM MgCl2, and 50 pmol of each primer. The cycling parameters were as follows: denaturation at 95°C for 45 s, annealing at 55°C or 58°C for 45 s, and extension at 72°C for 45 s for 35 cycles, followed by a final extension at 72°C for 7 min. PCR products were visualized in a 1% agarose gel by ethidium bromide staining.
For quantification of ARG1, FIZZ1/RELM, IL-4, IL-5, IL-13, IL-21, iNOS, tissue inhibitor of metalloproteinase-1 (TIMP-1), matrix metalloproteinase-12 (MMP-12), fibronectin, factor XIIIA (FXIIIA), and CCR4 mRNA, real-time RT-PCR was performed on a Chromo4 real-time PCR machine (Genetic Research Instrumentation Ltd., UK) using Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen). The following primers were used: ARG1 forward: 5'-CAG AAG AAT GGA AGA GTC AG-3', ARG1 reverse: 5'-CAG ATA TGC AGG GAG TCA CC-3'; FIZZ1 forward: 5'-GGT CCC AGT GCA TAT GGA TGA GAC CAT AGA-3', FIZZ1 reverse: 5'-CAC CTC TTC ACT CGA GGG ACA GTT GGC AGC-3'; IL-4 forward: 5'-ACA GGA GAA GGG ACG CCA T-3', IL-4 reverse: 5'-GAA GCC CTA CAG ACG AGC TCA-3'; IL-5 forward: 5'-CAA TGA GAC GAT GAG GCT TCC TG-3', IL-5 reverse: 5'-ACC CCC ACG GAC AGT TTG ATT-3'; IL-13 forward: 5'-AGA CCA GAC TCC CCT GTG CA-3', IL-13 reverse: 5'-TGG GTC CTG TAG ATG GCA TTG-3'; IL-21 forward: 5'-GCC AGA TCG CCT CCT GAT TA-3', IL-21 reverse: 5'-CAT GCT CAC AGT GCC CCT TT-3'; iNOS forward: 5'-GCA TTT GGG AAT GTA GAC T-3'; iNOS reverse: 5'-GTT GCA TTG GAA GTG AAG GGT TT-3'; TIMP-1 forward: 5'-GTG GGA AAT GCC GCA GAT-3'; TIMP-1 reverse: 5'-GGG CAT ATC CAC AGA GGC TTT-3'; MMP-12 forward: 5'-CAA TTG GAA TAT GAC CCC CTG T-3', MMP-12 reverse: 5'-AGC AAG CAC CCT TCA CTA CAT-3'; fibronectin forward: 5'-AAT GGA AAA GGG GAA TGG AC-3', fibronectin reverse: 5'-CTC GGT TGT CCT TCT TGC TC-3'; FXIIIA forward: 5'-GTT GCA GTC TGG ACT CCC TA-3', FXIIIA reverse: 5'-TAC ACA GCG TCC TCT TCA CA-3'; CCR4 forward: 5'-TCT ACA GCG GCA TCT TCT TCA T-3', CCR4 reverse: 5'-CAG TAC GTG TGG TTG TGC TCT G-3'; β-actin forward: 5'-TGG AAT CCT GTG GCA TCC ATG AAA C-3', β-actin reverse: 5'-TAA AAC GCA GCT CAG TAA CAG TCC G-3'. The PCR reaction was carried out in 10.5 µl containing 1 µl cDNA, 0.3 mM primers, and SYBR Green Supermix-UDG. The amplification was performed under the following conditions: 15 min hot start at 95°C, followed by 50 cycles of denaturation at 95°C for 20 s, primer annealing at 55°C for 20 s, and an elongation step at 72°C for 20 s. The fluorescent DNA-binding dye SYBR Green was monitored after the elongation step of each cycle for 1 s at 80°C. To identify and determine the purity of the reaction products after the DNA amplification, a melting curve was generated by raising the temperature from 55°C to 95°C and reading the fluorescence 2 s after every 1°C increase in temperature. For each gene, five serial 1:4 dilutions of cDNA of a positive control sample were used in each reaction. Expression levels were normalized against the housekeeping gene β-actin.
Statistical analysis
Graphpad Prism 4 software (Graph Pad Software, San Diego, CA, USA) was used for the statistical analysis of the real-time RT-PCR data. The two-tailed Mann-Whitney nonparametric t-test was used to assess the statistical difference between groups, and P < 0.05 was designated as significant.
RESULTS
Migration of splenic Ms to the sites of latent infection in IFN-R–/– mice
Situated throughout the spleen are different subsets of Ms, the majority of which reside in the red pulp. The red pulp Ms are detectable using the F4/80 mAb that recognizes a murine M-restricted, cell-surface glycoprotein with homology to the G-protein-linked transmembrane 7 hormone receptor family [38
]. Ms are embedded in a fine reticulin meshwork and for the most part, form a syncitial network of interconnecting cell processes. We studied the distribution of F4/80-positive (F4/80+) Ms in the spleens of wild-type and IFN-R–/– mice at different times post-MHV-68 infection. Up to Day 8 p.i., F4/80+ Ms were distributed in the red pulp of spleens of wild-type and IFN-R–/–. However, at Day 12, the red pulp Ms displayed a morphologically activated phenotype, and "amoeboid-like" F4/80+ Ms started to migrate into the GC in IFN-R–/– mice (Fig. 1A
). The F4/80+ Ms were visible in the GC of IFN-R–/– mice until Day 16 p.i., after which their numbers declined. By Day 20, only isolated F4/80+ cells were detected in the GC. At this time, the spleens of IFN-R–/– mice contain large deposits of collagen, in marked contrast to spleens in wild-type mice, where small amounts of collagen are detected (Fig. 1B)
. By Day 35, when fibrosis is resolved, and the spleen resumes a normal structure, the distribution of F4/80+ cells in the IFN-R–/– mice resembles that seen in wild-type, infected mice (Fig. 1A)
. Thus, MHV-68 infection of IFN-R–/– mice but not wild-type mice leads to the activation of F4/80+ Ms, resulting in their migration from the red pulp to the GCs of the spleen, where latent virus-infected B cells are located [5
6
7
8
].
 View larger version (90K): [in a new window] |
Figure 1. Kinetics and distribution of F4/80-positive Ms and extent of fibrosis in the spleen of MHV-68-infected IFN-R–/– and wild-type mice. (A) Sections of spleens harvested at Days 8, 12, 16, 20, and 35 p.i. from MHV-68-infected wild-type and IFN-R–/– mice were stained with F4/80, which stains red pulp (RP) Ms. In wild-type mice, F4/80+ Ms are confined to the red pulp throughout the course of infection. In IFN-R–/– mice, F4/80+ cells are found in the red pulp at Day 8 but migrate into the GCs at Days 12 and 16. At Day 20, when there is considerable fibrosis, F4/80+ cells are scattered throughout red pulp and fibrotic areas, and a few cells remain in the GCs. By Day 35, when fibrosis is resolved, the distribution of F4/80+ cells is similar to that in wild-type mice. C, Collagen. (B) Picrosirius Red staining showing fibrosis in IFN-R–/– mice but not wild-type mice at Day 20 p.i. Original magnification: Days 8 and 35, 10x; Days 12, 16, and 20, 20x. Photographs are representative examples of two to three experiments with three to five mice.
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s in the spleen show different functional properties and distribution patterns [39
]. A specific subset of Ms in the marginal zone of the spleen is positive for a mAb, called ER-TR9, and this antibody targets the specific ICAM3-grabbing nonintegrin (SIGN)-R1 antigen, which is one of the five recently identified mouse genes that are homologous to a human DC-SIGN [40
]. Immunohistochemical analysis of spleens, taken from IFN-R–/–-infected mice at Day 12 p.i., showed an increase in the number of ER-TR9-positive cells in the marginal zone area migrating toward the GC (Fig. 2
). Marginal metallophils are positioned in the inner side of the marginal zone in close proximity to white pulp and identified by MOMA-1 antibodies. MOMA-1-positive marginal metallophils were also observed in increased numbers at Day 12 p.i. Like the ER-TR9-positive cells, MOMA-1-positive cells had also migrated into the GC (Fig. 2)
. The pattern of migration of ER-TR9- and MOMA-1-positive Ms follows the same kinetics as the F4/80 Ms (data not shown). Taken together, our data show that in MHV-68-infected IFN-R–/– mice, the distribution of three subpopulations of splenic Ms changes from their normal, anatomical location in the spleen and Ms migrate to GCs, where the latently infected cells are located. These events are not seen in MHV-68-infected, wild-type mice.
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Figure 2. Spleens of infected wild-type and IFN-R–/– mice show different histological profiles of marginal zone (MZ) and marginal metallophilic Ms. The presence of marginal zone Ms and marginal metallophils was analyzed in spleens taken from infected wild-type and IFN-R–/– mice by immunohistochemical staining with ER-TR9 and MOMA-1, respectively. Ms of both subsets increased in number and showed an activated phenotype and movement toward the GCs in infected IFN-R–/– mice at Day 12 p.i. In contrast, these Ms were only present in low numbers in the marginal zone in infected wild-type mice. Original magnification, 20x. Photographs are representative examples of two to three experiments with three to five mice.
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and tissue remodeling occur in the spleens of IFN-R–/– mice infected with MHV-68s in the GC of spleens from MHV-68-infected IFN-R–/– mice suggests these cells contribute to the evolving fibrosis. In the absence of a functional IFN- response, we hypothesized that the immune response is skewed toward a type-2 cytokine environment favoring the alternative activation of Ms. Well-characterized markers for aaMs include ARG1 and FIZZ1/RELM [21
, 23
24
25
]. We therefore analyzed expression of these markers in the spleen at various times p.i. by RT-PCR (data not shown), real-time RT-PCR, and/or immunohistochemistry.
Real-time RT-PCR revealed increased levels of ARG1 and FIZZ1 in infected IFN-R–/– mice compared with wild-type mice (P<0.05) in a time-dependent manner (Fig. 3A
). Expression of ARG1 increased during active development of fibrosis on Days 12, 16, and 20 p.i. and had returned to basal levels by Day 35 (Fig. 3A)
. Likewise, expression of FIZZ1 increased during development of fibrosis, being detected on Days 16 and 20 p.i., and had returned to basal levels by Day 35 (Fig. 3A)
. In contrast, expression of iNOS, a marker of classically activated Ms, increased in spleens from infected wild-type mice but not IFN-R–/– mice (P<0.05; Fig. 3A
). Taken together, these data support the development of aaMs in spleens from herpesvirus-infected IFN-R–/– mice.
 View larger version (24K): [in a new window] |
Figure 3. Spleens from MHV-68-infected IFN-R–/– mice but not wild-type mice show increased expression of markers of aaM and tissue remodeling. Quantitative RT-PCR analysis of RNA isolated from spleens of MHV-68-infected wild-type and IFN-R–/– mice, harvested at various time-points postinfection. mRNA expression, expressed as fold increase over β-actin of ARG1 and FIZZ1 (A) and TIMP-1, MMP-12, fibronectin, and FXIIIA (B), is significantly higher in the infected IFN-R–/– mice compared with the wild-type mice (P<0.05), and iNOS expression only increased in spleens of infected, wild-type mice (P<0.05; A). Graphs are representative examples of two to three experiments with three to five mice. *, P < 0.05.
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s and analyze their distribution, sections of spleen were stained for ARG1. ARG1-positive cells were detected in the GCs of infected IFN-R–/– mice, and no staining was detected in the spleens from wild-type mice (Fig. 4A
). ARG1 expression colocalized with different subsets of Ms (F4/80, ER-TR9, and MOMA-1) in the GCs of spleens of infected IFN-R–/– mice (Day 12 p.i.; Fig. 4B
).
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Figure 4. M subsets in GC of spleens from MHV-68-infected IFN-R–/– mice express ARG1. (A) Sections of spleens harvested at Day 12 p.i. from MHV-68-infected wild-type and IFN-R–/– were stained with an antibody against ARG1. ARG1-positive cells were only detected in spleens of IFN-R–/–mice. Original magnification, 20x. (B) ARG1-positive cells in spleens of MHV-68-infected IFN-R–/– mice (Day 12 p.i.) also express F4/80, ET-TR9, and MOMA-1. Figure
shows negative controls for ARG1 staining and for ER-TR9 M staining. NRatS, normal rat serum; NRbS, normal rabbit serum. Original magnification, 90x. Photographs are representative examples of two to three experiments with three to five mice.
|
have various profibrotic properties [21
, 29
, 41
, 42
] directly linking the development of aaMs with fibrogenesis seen in spleens of herpesvirus-infected IFN-R–/– mice. To address this profibrotic role of the Ms in more detail, we analyzed the expression of mediators involved in extracellular matrix (ECM) turnover. The pattern of ECM turnover is regulated by TIMPs that hold in check the activation of concurrently secreted MMPs. After performing a microarray analysis to study the expression of a panel of MMPs and TIMPs, we saw specific up-regulation of TIMP-1 and MMP-12 in spleens from infected IFN-R–/– mice only at the time of fibrosis (data not shown). Using quantitative RT-PCR, we confirmed this increase (P<0.05; Fig. 3B
).
Monocytes contribute to ECM remodeling, not only by degrading its proteins via proteases but also by depositing ECM proteins, including fibronectin, which assembles through cross-linking into fibrils attaching cells to the ECM and is important for cellular migration during development and wound healing. Ms secrete, bind, and migrate in response to fibronectin [43
44
45
46
]. Moreover, fibronectin promotes the differentiation of monocytes into tissue Ms [45
]. Quantitative RT-PCR of fibronectin showed highly increased expression of this protein in spleens of infected IFN-R–/– mice but not wild-type mice at the height of fibrosis (P<0.05; Fig. 3B
). The fibronectin cross-linking protein FXIIIA, which has recently been described as a marker of aaM [47
, 48
], also showed a pronounced up-regulation in spleens of infected IFN-R–/– mice only (P<0.05; Fig. 3B
). Taken together, these findings underline development of aaMs and active ECM remodeling in fibrotic spleens of herpesvirus-infected IFN-R–/– mice.
Th2 cytokines are up-regulated in the spleen during alternative activation of Ms and induction of fibrosis
aaMs are the result of the activity of a number of Th2-derived cytokines [21
, 23
24
25
]. IL-4 and IL-13 are the principal activators of aaMs, but their function is augmented by IL-21 [49
]. Similarly, IL-5 polarizes the Th2 response and is required for development of IL-13-triggered responses [29
]. To assess the role of these cytokines in the activation of splenic Ms in our fibrosis model quantitative RT-PCR was carried out on spleen-derived RNA. In addition, expression of the chemokine receptor CCR4, a recognized, Th2 cell-associated marker [50
, 51
], was investigated. The expression of IL-13 showed a time-dependent up-regulation from Day 14 p.i. to Day 28 p.i. in IFN-R–/– mice (Fig. 5
; P<0.05). By Day 35, IL-13 expression had returned to basal levels. Similarly, transcript levels of IL-5, IL-21, and CCR4 were increased in IFN-R–/– mice, and peak levels of transcript occurred at Day 20 (Fig. 5
; P<0.05), coinciding with the peaks of ARG1 and FIZZ-1. Levels of IL-4 expression in IFN-R–/– mice were higher overall than in wild-type mice (P<0.05), but unlike the other cytokines and the alternative activation markers, the expression patterns of IFN-R–/– and wild-type mice were similar. Results were confirmed in an independent experiment. Thus, our findings indicate that MHV-68 infection of IFN-R–/– mice induces development of a Th2 response, which will drive alternative activation of Ms.
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Figure 5. Increased transcription of Th2 cytokines and CCR4 in spleens of MHV-68-infected IFN-R–/– mice. Quantitative RT-PCR analysis of RNA isolated from spleens of infected wild-type and IFN-R–/– mice, harvested at Days 14, 20, 28, and 35. Spleens of IFN-R–/– mice show increased IL-13, IL-4, IL-21, IL-5, and CCR4 mRNA expression (expressed as fold increase over β-actin) compared with wild-type mice (P<0.05). Graphs are representative examples of two to three experiments with three to five mice.
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Infection of IFN-R–/– mice with MHV-68 results in a fibrotic pathology in various organs populated by latently infected cells. It is most pronounced in the spleen, where a generalized fibrosis occurs, leading to disruption of tissue architecture, a dramatic loss of leukocytes, and a blockade of leukocyte traffic entering the spleen [13
]. Remarkably, the whole process resolves by Day 35 p.i., and the spleen resumes normal function. In this paper, we explore the cellular events centring on latently infected GCs, in particular, the role of activated Ms in the different phases of splenic fibrosis.
In MHV-68-infected IFN-R–/– mice, activated Ms were populating the GC in the spleen by Day 12 p.i. Various M subsets were present, including F4/80+ cells (normally resident in the red pulp), ER-TR9-positive cells (normally resident in the marginal zone), and MOMA-1-positive cells (normally resident on the inner side of the marginal zone). These cells clearly migrate into the GC in infected IFN-R–/– mice, where they express ARG1. To our knowledge, this is the first demonstration of distinct M subsets expressing ARG1 during the evolution of fibrosis.
The recruitment of different M subsets into the GC is presumably the result of a T cell-mediated response against latently infected cells. What role is played by the individual M subsets during this phase is unclear and the subject of ongoing research in our laboratory. The M subsets are still present in GCs at Day 16 but disappear by Day 20 as collagen deposition intensifies. This disappearance coincides with the mass exodus of leukocytes from the spleen and a blockade of cell trafficking from the bloodstream [13
]. Three weeks p.i., at the height of fibrosis, only isolated Ms are observed (Fig. 1A)
. The dominant cell type present at Days 20–30 is the fibroblast. One possible explanation for the sudden loss of Ms between Days 16 and 20 is that they transdifferentiate into fibroblasts/myofibroblasts [52
53
54
]. Resolution of fibrosis occurs by Day 35 with a repopulation of the spleen by F4/80+ Ms (Fig. 1A)
and B and T cells (data not shown).
In IFN-R–/– mice infected with MHV-68, splenic Ms become activated via the alternative pathway and recruited to the site of infection. These aaMs lack expression of iNOS, in contrast to the classically activated Ms in wild-type, infected mice, but instead, produce ARG1 and FIZZ1, as well as fibronectin, TIMP-1, MMP-12, and FXIIIA proteins that have been implicated in tissue repair and ECM turnover [29
, 30
, 41
, 55
] resembling the human IL-4-induced aaMs described by Gratchev et al. [46
, 48
]. The transient expression of these aaM markers in the IFN-R–/– mice corresponds with collagen deposition and widespread fibrosis in the spleen and shutdown of cell trafficking into and out of the tissue [13
] and underline a role for aaMs in tissue remodeling. Further, in the absence of IFN- signaling, the classical activation of Ms is absent and hence, the regulation of fibrogenesis by cytokines and growth factors that modulate the proliferation of and collagen synthesis by fibroblasts defective [56
].
In addition to a role in tissue remodeling through the induction of collagen deposition, ARG1 has been implicated as an antiviral agent and a regulator of T cell proliferation. In a productive herpesvirus infection, arginine is an essential amino acid for virus growth. Depletion of arginine by ARG1 aborts a productive infection, thereby providing an antiviral defense mechanism [57
]. Moreover, extracellular arginine depletion by ARG1 has been shown to influence the function of T cells. M activation that leads to ARG1 production will result in a rapid depletion of extracellular levels of arginine compared with products of other metabolic pathways [58
]. Thus, it is possible that ARG1 production by Ms in the GC acts as a natural control mechanism to regulate the activation of T lymphocytes, thereby limiting further immunopathology.
By Day 35 p.i., a time-point corresponding to the resolution of fibrosis and recovery of spleen architecture and function, ARG1, FIZZ1, TIMP-1, MMP-12, fibronectin, and FXIIIA expression is no longer evident. By this time, latently infected cells are difficult to detect, suggesting that these have been purged from the GC (data not shown). We also investigated the aaM marker Ym1 [21
, 23
, 24
] but found no consistent pattern of expression (data not shown). Taken together, our data from the spleen are consistent with the MHV-68-induced chronic pulmonary fibrosis model described by Mora et al. [14
], in which there is early up-regulation of FIZZ1 and ARG1 at the height of lung pathology.
The concept of an aaM pathway by the Th2-type cytokines IL-4 and IL-13 has gained credence in the past decade to account for a distinctive M phenotype involved in tissue repair [21
]. These interleukins have broadly similar effects on Ms, and they share a common receptor chain. IL-4 and IL-13 promote ARG1-dependent formation of L-ornithine, which can be converted to polyamines and proline, important for cellular proliferation and collagen production, respectively [28
]. These processes can be inhibited by regulating the expression of IL-13 using blocking antibodies, soluble receptors, or chimeric proteins [59
], demonstrating a crucial role for this cytokine in tissue remodeling. Here, we show that IL-13 expression is induced in the spleen of MHV-68-infected IFN-R–/– mice around Days 12–20. These kinetics correspond with the induction of ARG1 and FIZZ1, markers for the aaMs, and the establishment of fibrosis in the spleen of infected IFN-R–/– mice. In addition to IL-13, we found elevated transcription of the Th2 cytokines IL-5 and IL-21 around Days 12–20. IL-5 has long been recognized as a product of Th2 cells and recently, has been shown to regulate fibrotic disease by modulating IL-13 production [29
]. IL-21 promotes the development of Th2 responses and Th2-dependent pathologies and augments the development of aaMs [49
, 60
]. Moreover, in the absence of the IL-21R, pathogen-induced fibrosis is reduced significantly [49
]. Together with the elevated transcription of IL-4 and of CCR4, a Th2-associated chemokine receptor, at Day 20, our data provide strong evidence for a Th2 bias in the spleens of MHV-68-infected IFN-R–/– mice. This observation correlates with the appearance of CD4+ and CD8+ T cells in the GC of spleens of infected mice (data not shown) and suggests Th2- and Tc2-type differentiation is actively involved in driving the immune response to a fibrotic pathology. The importance of T cells in this process is highlighted by depletion of CD4 or CD8 T cells preventing fibrosis from occurring [11
].
Our observations may have broader implications for the genesis of fibrotic lesions in man. Many fibrotic disorders are of unknown etiology, but herpesviruses represent good candidates for initiating these pathological events as a result of their persistent nature. Indeed, a potential link exists between EBV and IPF, where viral DNA and lytic cycle viral antigens (immunocytochemistry) were detected in 
50% of IPF patients [61
, 62
]. The model described here provides a tool for studying the etiology of fibrotic disease.
In summary, although the detailed mechanisms underlying evolution and resolution of fibrosis have yet to be resolved, based on our findings, we hypothesize that the development of aaMs in response to Th2-derived cytokines and CCR4, which we show are induced 2–3 weeks p.i., leads to fibrosis through M-specific products including ARG1 and FIZZ1/RELM. As a result, the latent virus is cleared and/or prevented from further reactivation. Consequently, the trigger for aaM disappears, resulting in their replacement by and/or switch to another M type. This leads to a reduction of the aaM-specific, profibrotic proteins, allowing for resolution of fibrosis to occur. These studies may have important implications for understanding how fibrotic disorders in humans can be resolved.
ACKNOWLEDGEMENTS
This work was supported by the Biotechnology and Biological Sciences Research Council and a Ter Meulen Fund fellowship (to M. A. H.). The authors thank Ian Bennet for excellent technical assistance.
FOOTNOTES
1 These authors contributed equally to this work. 
2 Current address: Roslin Institute, Neuropathogenesis Unit, Edinburgh, EH9 3JF, UK.
Received May 2, 2007; revised March 12, 2008; accepted March 13, 2008.
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