TLR9-induced interferon β is associated with protection from gammaherpesvirus-induced exacerbation of lung fibrosis
© Luckhardt et al; licensee BioMed Central Ltd. 2011
Received: 7 March 2011
Accepted: 2 August 2011
Published: 2 August 2011
We have shown previously that murine gammaherpesvirus 68 (γHV68) infection exacerbates established pulmonary fibrosis. Because Toll-like receptor (TLR)-9 may be important in controlling the immune response to γHV68 infection, we examined how TLR-9 signaling effects exacerbation of fibrosis in response to viral infection, using models of bleomycin- and fluorescein isothiocyanate-induced pulmonary fibrosis in wild-type (Balb/c) and TLR-9-/- mice.
We found that in the absence of TLR-9 signaling, there was a significant increase in collagen deposition following viral exacerbation of fibrosis. This was not associated with increased viral load in TLR-9-/- mice or with major alterations in T helper (Th)1 and Th2 cytokines. We examined alveolar epithelial-cell apoptosis in both strains, but this could not explain the altered fibrotic outcomes. As expected, TLR-9-/- mice had a defect in the production of interferon (IFN)-β after viral infection. Balb/c fibroblasts infected with γHV68 in vitro produced more IFN-β than did infected TLR-9-/- fibroblasts. Accordingly, in vitro infection of Balb/c fibroblasts resulted in reduced proliferation rates whereas infection of TLR-9-/- fibroblasts did not. Finally, therapeutic administration of CpG oligodeoxynucleotides ameliorated bleomycin-induced fibrosis in wild-type mice.
These results show a protective role for TLR-9 signaling in murine models of lung fibrosis, and highlight differences in the biology of TLR-9 between mice and humans.
Idiopathic pulmonary fibrosis (IPF) is a progressive, fibrotic lung disease with a poor survival rate. Causes of IPF may relate to epithelial-cell injury, abnormal fibroproliferation, inflammation, and deposition of extracellular matrix components [1, 2]. Standard therapies have shown little benefit, and most patients progress to respiratory failure.
Many patients with IPF have a slow progressive disease course over months to years after diagnosis ; however, some patients experience acute deterioration in pulmonary function [3–7] without clear cause. This is referred to as acute exacerbation of IPF. Histological findings are diffuse alveolar damage or organizing pneumonia plus usual interstitial pneumonitis [4–7]. Reported mortality rates are often greater than 78% in this population of patients .
Viral infections have been linked to the development of fibrosis in both human and animal studies [8–13]. We have shown previously that γHV68 infection augments fluorescein isothiocyanate (FITC)-induced pulmonary fibrosis when given 14 days after fibrotic challenge . This exacerbation of fibrosis caused by γHV68 shares many similarities with acute exacerbations in humans, including decreased lung compliance and diffuse alveolar damage on histopathology .
The pathogenesis of IPF is unclear. Many cell types have been implicated in the pathogenesis of the fibrotic response, including mesenchymal cells [15–17] and alveolar epithelial cells (AECs) . AECs are closely approximated to the mesenchymal (fibroblast) cells within the lung, and are believed to play an important role in homeostasis between epithelial and mesenchymal cells. AEC injury is a universal feature seen in the pathobiology of fibrotic lung disease , and there is good evidence that AEC apoptosis plays a role in the pathogenesis of pulmonary fibrosis [20–22]). A role for gammaherpesviral infection in the induction of AEC apoptosis has been suggested. Egan et al. reported that, compared with controls, patients with IPF had increased Epstein-Barr virus (EBV) DNA loads in lung tissue, and some of these patients also had positive staining for p53, suggesting an increase in apoptosis . Additionally, in vitro infection of human lung epithelial cells with EBV induced secretion of transforming growth factor (TGF)-β1 secretion and upregulated caspases 3 and 7 . However, mechanistic studies using animal models have not been carried out.
The human immune system has a series of 10 innate immune receptors known as Toll-like receptors (TLRs), which enable host cells to recognize foreign pathogens. TLR-9 is an endosomal receptor that recognizes unmethylated CpG nucleotides, which are commonly found in bacterial and viral DNA genomes . Stimulation of TLR-9 results in activation of the MyD88 pathway, resulting ultimately in the activation of Jun N-terminal kinase and the translocation of nuclear factor (NF)κB to the nucleus . TLR-9 stimulation is associated with the development of T helper (Th)1 immune responses [25, 26]. TLR-9 is expressed at its highest levels in plasmacytoid dendritic cells (DCs) and B cells , but it has also been found on lung epithelial cells  and fibroblasts . TLR-9 has been shown to be important in the pathogenesis of γHV68, with TLR-9-/- mice being more susceptible to both lytic and latent γHV68 infection after intraperitoneal inoculation .
In this paper, we show that TLR-9 signaling can limit the exacerbation of the fibrotic response by γHV68 infection in the lung. This exacerbation is not associated with increased epithelial-cell apoptosis or major alterations in Th1 or Th2 cytokines, but rather, viral infection in wild-type, but not TLR-9-/- mice, leads to production of IFN-β and diminished fibroproliferation. Furthermore, therapeutic administration of CpG oligodeoxynucleotides (ODN) can limit bleomycin-induced pulmonary fibrosis.
Toll-like receptor-9 signaling protects mice from viral-induced exacerbation of fibrosis, but has no effect on fibrotic insults alone
Fibrotic mice are more susceptible to viral replication, but Toll-like receptor-9 deficiency does not alter replication in the lungs
Alveolar epithelial cells, fibroblasts and fibrocytes all express Toll-like receptor-9
Apoptosis of alveolar epithelial cells does not differ between Balb/c and TLR9-/- mice treated with bleomycin and gammaherpesvirus 68
AECs were also isolated from naïve Balb/c or TLR-9-/- mice, and these cells were infected with either 0.01 or 0.001 MOI (multiplicity of infection) of γHV68 for 48 hours, before cell lysates were prepared and analyzed for cleaved caspase 3 by western blotting. Viral infection induced caspase 3 activation in both genotypes, but there was no evidence of increased caspase 3 activation in the TLR-9-/- cells compared with the Balb/c cells (Figure 4C). Thus, the reason TLR-9-/- mice develop worse fibrosis does not appear to be related to alterations in AEC apoptosis.
Balb/c and TLR9-/- alveolar epithelial cells are equivalent in their ability to suppress fibroproliferation
To determine whether there was an inherent difference in the ability of AECs from Balb/c and TLR-9-/- mice to limit fibroproliferation, AECs were isolated from either Balb/c or TLR-9-/- mice, and these cells were co-cultured with fibroblasts from either Balb/c mice (see Additional file 1, supplementary Figure 3A) or with fibroblasts from TLR-9-/- mice (see Additional file 1, supplementary Figure 3B). AECs from both strains were equivalent in their ability to limit fibroproliferation over a 24 h period.
TLR9-/- mice treated with bleomycin plus gammaherpesvirus 68 show increased CD8 recruitment
In our previous work, we found that viral exacerbation of fibrosis in wild-type mice correlated with viral-induced fibrocyte accumulation in the lung . To determine whether fibrocyte recruitment was different between Balb/c and TLR-9-/- mice treated with bleomycin plus γHV68, lung leukocytes were isolated by collagenase and DNAse digestion on day 21, and these cells were stained for CD45+ collagen 1+ cells. Lungs of Balb/c mice contained 1.2 ± 0.14% fibrocytes, which was not significantly different from the 2.02 ± 0.5% seen in TLR-9-/- mice (n = 4, P = NS). Thus, differential recruitment of fibrocytes was not seen in TLR-9-/- versus control mice.
TLR9-/- mice have reduced T helper 2 responses during viral exacerbation of fibrosis
Balb/c mice and TLR-9-/- mice were treated with bleomycin on day 0. On day 14 the mice were given γHV68 infection (5 × 104 PFU). On days 0, 14, 17 and 21, lungs were harvested, and whole-lung homogenates were assayed for Th1 and Th2 cytokines by ELISA. Because γHV68 has been shown to be fibrotic in Th2-biased mice , we wanted to determine whether TLR-9-/- mice had increased Th2 profiles. There were modest fluctuations in the Th2 cytokines over the 21 day course, but no evidence of a Th2 skewing in the TLR-9-/- mice (see Additional file 1, supplementary Figure 4A, B). In fact, when treated with bleomycin plus γHV68, the TLR-9-/- mice had decreased levels of interleuking (IL)-4 and IL-13 on day 21 compared with baseline measurements; these trends were not noted in the Balb/c mice. When Th1 cytokines were analyzed, both Balb/c and TLR-9-/- mice mounted strong interferon-γ responses by day 21 of the experiment, which corresponded to day 7 after viral infection (see Additional file 1, supplementary Figure 4(C). By contrast, IL-12 levels diminished after infection in both genotypes (see Additional file 1, supplementary Figure 4(D). There were few changes in expression profiles of IL-17 or tumor necrosis factor-α (see Additional file 1 supplementary Figure 4E and 4F). Additionally, when total TGF-β levels were measured after acid activation by ELISA on day 21 following bleomycin plus γHV68 infection, there was no significant difference between the genotypes (see Additional file 1, supplementary Figure 5).
TLR9-/- mice are defective in IFN-β expression post challenge
Gammaherpesvirus 68 infection induces IFN-β production, which limits fibroblast proliferation
Gammaherpesvirus 68 infection reduces proliferation of Balb/c, but not TLR9-/- fibroblasts
CpG therapy can limit bleomycin-induced fibrosis
We have previously shown that a gammaherpesvirus infection can exacerbate established FITC-induced fibrosis in a murine model . In this study, we found that TLR-9 signaling plays a role in limiting the profibrotic exacerbations of gammaherpesviral infection. There are many potential mechanisms by which TLR-9 signaling might influence the fibrotic environment during viral infection. One possible mechanism is simply by limiting viral replication. Guggemoos et al. showed that TLR-9 signaling is important in control of γHV68 infection when given intraperitoneally ; however, they were not able to show that TLR-9 signaling in the lung was important in controlling an intranasal infection with γHV68. Our data are consistent with this finding. Although replication of the virus was greater in mice treated with bleomycin compared with non-fibrotic mice, the absence of TLR-9 had little effect on viral gene expression in either group. Although it is not entirely clear why viral replication is enhanced in fibrotic mice, it is likely that proinflammatory and profibrotic factors enhance viral gene transcription. In support of this hypothesis, prostaglandin E2 has been shown to promote viral replication . Thus, our results confirm that TLR-9 signaling is not required for control of lytic γHV68 infection in the lung. The reasons why TLR-9 would differentially regulate lung versus peritoneal infection probably reflect the differences in the cell populations that are initially infected in each site.
Within the lung, AECs are one of the primary cell types infected with γHV68, as confirmed by our immunohistochemical findings in this study (Figure 4A) and our previous studies [31, 32]. Although it is known that γHV68 expresses proteins that can prevent apoptosis and enable establishment of latent infection [37–39], previous studies in human cells and tissues have suggested a role for gammaherpesviruses in the induction of AEC apoptosis [10, 23]. Furthermore, TLR-9 stimulation has been shown to inhibit macrophage apoptosis . Thus, we were surprised to discover that levels of apoptosis in AECs isolated from mice treated with bleomycin plusγHV68 were similar between Balb/c and TLR-9-/- genotypes. This was true regardless of whether apoptosis was assessed by M30 staining or caspase activation.
One important aspect of AEC function is to limit fibroproliferation. Profibrotic stimuli (for example, chemokine (C-C motif) ligand 2 (CCL2)) are known to alter the ability of isolated AECs to limit fibroproliferation . Thus, we sought to determine whether there were basal differences in the ability of the AECs from Balb/c or TLR-9-/- mice to control fibroblast proliferation. However, AECs from both genotypes of mice were equivalent in their ability to limit fibroblast proliferation from both strains. Taken together, we could not find substantial evidence of altered AEC apoptosis or function to explain the exaggerated viral-induced fibrotic response noted in TLR-9-/- mice.
We next investigated the inflammatory response between Balb/c and TLR-9-/- mice treated with bleomycin plus γHV68. After viral exacerbation, both genotypes of mice had an increase in total numbers of inflammatory cells, but there were few differences noted in the particular cell types recruited. There were no significant differences in fibrocyte accumulation, or in the percentages of neutrophils or CD4, natural killer or B cells between genotypes. The TLR-9-/- mice did have a higher percentage of CD8+ cells, and it is possible that if activated, these CD8+ cells could contribute to tissue damage, which might exaggerate the fibrotic response in the TLR-9-/- mice. Both genotypes had a loss of B cells after infection, and we believe this represents the migration of B cells to the spleen, the known major reservoir for latent viral infection .
Because TLR-9 signaling leads to NFκB activation and the production of Th1 cytokines , it seemed likely that TLR-9-/- mice would have a more Th2-biased cytokine environment. Because Th2-biased mice are known to be prone to the development of fibrosis in response to infection with γHV68 , it was possible that a cytokine imbalance could explain the exacerbation of fibrosis in TLR-9-/- mice. However, there was no evidence of Th2 skewing or defective Th1 signaling in the TLR-9-/- mice during the acute response to bleomycin and infection. In fact, IL-4 and IL-13 levels were reduced on day 21 after bleomycin plus γHV68 administration in TLR-9-/- mice. We were surprised that IFN-γ was not diminished in the TLR-9-/- mice, and we believe that this may reflect the fact that in AECs at least, activation of other TLRs may help to stimulate NFκB activation for IFN-γ production. We confirmed that lung AECs from TLR-9-/- mice express normal levels of TLR7 and TLR8, for instance (data not shown). Additionally, differences in production of TGF-β were not noted between genotypes. The observation that fibrosis is exaggerated despite strong induction of IFN-γ confirms our previous findings of viral exacerbation in the C57Bl/6 background . Thus, we conclude that neither reduced production of Th1 cytokines nor increased production of Th2 cytokines in response to infection can explain the enhanced exacerbation of fibrosis noted in TLR-9-/- mice.
Not surprisingly, the one cytokine whose production was different between Balb/c and TLR-9-/- mice was IFN-β. TLR-9 stimulation is known to induce type I interferons . Levels of IFN-β were reduced after bleomycin plus virus in whole-lung homogenates and in infected fibroblasts. It is interesting that after bleomycin administration alone, levels of IFN-β were reduced (Figure 6A). The fact that this happened in both Balb/c and TLR-9-/- mice suggest a TLR-9-indepdent mechanism for the reduction in IFN-β after a fibrotic insult. This should not necessarily be interpreted to mean that bleomycin causes a reduction in TLR-9 expression, as we have no evidence of that, at least in fibroblasts isolated from fibrotic mice (data not shown). As IFN-β is known to be a potent inhibitor of lung fibroblast proliferation , a fact we have confirmed (Figure 7), it is reasonable to conclude that early viral infection of fibroblasts would result in the stimulation of TLR-9 by viral CpG DNA sequences and a concomitant decrease in fibroblast proliferation. In fact, we found that fibroblasts from Balb/c mice potently upregulated IFN-β secretion in response to even low-level infection. Furthermore, exogenous IFN-β could limit proliferation of fibroblast from both strains, suggesting that the defect in TLR-9-/- mice is not sensitivity to, but rather production of, IFN-β. Taken together, we conclude that although viral infection after fibrotic challenge increases the fibrotic response of both strains, the more extensive exacerbation of fibrosis noted in the TLR-9-/- mice is most likely due to deficiencies in IFN-β production, which in turn allow for increased fibroproliferation. When cell number and viral dose were equivalent, fibroproliferation was inhibited in Balb/c, but not TLR-9-/- mice (Figure 8). Because there may be some TLR-9-independent signaling that leads to IFN-β production in the lung by day 7 after infection (Figure 6A), it is possible that at later time points, sufficient IFN-β may be available to limit fibroproliferation even in TLR-9-/- mice. It is important to remember that secretion of IFN-β in the lung would probably inhibit the proliferation of other resident fibroblasts, not just those that happened to be infected.
TLR-9-/- mice did not respond differently from wild-type mice to bleomycin alone or FITC alone. These results suggest that fibrotic insults alone may not generate endogenous ligands for TLR-9 stimulation. Thus, we next wanted to determine whether therapeutic stimulation of TLR-9 with CpG ODN could protect against fibrotic challenge. We initially tried to perform these experiments in the Balb/c background, but had poor responses to bleomycin in two separate experiments. Although levels of collagen were somewhat lower in CpG ODN-treated mice than in control mice, the overall levels of fibrosis were so low that meaningful interpretations of the data were not possible. Given the expense of these experiments, we chose to perform them in the C57Bl/6 background, which has a more reproducible fibrotic response to bleomycin or Blenoxane . We found that mice treated intranasally with CpG ODN were significantly protected from the development of bleomycin-induced fibrosis (Figure 9). Although these results in murine studies were very exciting, recent studies in human cells have made it clear that it is unlikely that these results can be extrapolated to humans.
Our results are not the first to describe an enhanced fibrotic response in TLR-9-/- mice. Earlier studies have shown that granulomas that form in response to Schistosoma mansoni eggs in TLR9-/- mice are larger and have more collagen deposition within the granuloma than in wild-type mice . However, in that study, the results were associated with diminished Th1 and augmented Th2 responses, probably reflecting the strong Th2-skewing nature of the S. mansoni egg antigens, whereas we used a Th1-inducing viral infection. Thus, there are now at least two examples of TLR-9 signaling showing a protective effect in lung-fibrosis models in mice. By contrast, Meneghin et al. found that TLR-9 expression in human lung fibroblasts promotes myofibroblast differentiation , and was anticipated to worsen fibrotic disease. This human study also found that TLR-9 was expressed at low levels in surgical lung biopsies of normal subjects, but was dramatically upregulated in the biopsies from patients with fibrotic lung diseases. These data suggest that TLR-9 expression might be low in normal human lung fibroblasts, whereas our studies suggest that murine lung fibroblasts express this receptor constitutively. In addition, TLR-9 expression in human lung epithelium is modest compared with that at other sites in the body . Studies in both human lung fibroblasts and human hepatic stellate cells have shown the ability of TLR-9 stimulation via CpG ODN to induce myofibroblast differentiation [28, 46]. Furthermore, expression of TLR-9 on fibroblasts obtained from initial surgical lung biopsies of patients with IPF can distinguish patients with rapid disease progression (TLR-9-positive) from patients with a slower disease course (TLR-9-negative) . Thus, it seems that in humans, TLR-9 stimulation may promote fibrotic reactions, whereas in mice, it may protect. Another important difference that may explain the improved outcomes in TLR-9-expressing mice is that mice have a larger repertoire of CpG-responsive hematopoietic cells. In mice, all DC subsets, B cells and macrophages respond to CpG ODN, whereas in humans, only plasmacytoid DCs and B cells respond . Thus, it is likely that production of IFN-β in response to TLR-9 stimulation is greater in the murine system than it would be in humans. Collectively, these studies identify important differences between mice and men, and thus suggest that CpG therapy, although beneficial in rodent models of fibrosis, is unlikely to be beneficial for human treatment of IPF.
TLR-9 signaling is protective during viral exacerbation of murine pulmonary fibrosis. This is probably due to increased levels of IFN-β, which limit fibroblast proliferation. There is no evidence that TLR-9 signaling during a viral exacerbation of pulmonary fibrosis alters alveolar epithelial-cell apoptosis. As TLR-9 signaling in human IPF fibroblasts appears to lead to a more profibrotic phenotype, these data highlight important differences between the human and mouse disease, and the limitations of our current animal models of pulmonary fibrosis.
The Animal Use Committee at the University of Michigan (Ann Arbor, MI, USA) approved all protocols and experiments described.
BALB/c and C57Bl/6 mice (Jackson Laboratories, Bar Harbor, ME, USA), aged 6 to 8 weeks old and matched for age and sex, were used. TLR-9-/- mice backcrossed to Balb/c (University of Michigan, East Lansing, MI, USA) have been described previously .
Fluoroscein isothiocyanate and bleomycin models of pulmonary fibrosis
Intratracheal (IT) inoculation of FITC (50 μl of a 2.8 mg/ml solution in saline) or bleomycin sulfate (0.035 per mouse in a 50 μl volume) (both from Sigma Chemical Co., St. Louis, MO, USA) was performed as described previously . In C57Bl/6 mice, clinical grade bleomycin sulfate (Blenoxane; Bristol-Myers Squibb, New York, NY, USA) was utilized.
Mice were anesthetized with ketamine and xylazine. Into 20 μl saline were suspended 5 ×104 PFU of γHV68 (American Type Culture Collection, Manassas, VA, USA), which were then delivered intranasally to each mouse. Sham infections consisted of intranasal delivery of 20 μl of saline.
Lung collagen measurements
Total lung collagen measurements were made as described previously , using the Sircol collagen dye-binding assay (Accurate, Westbury, NY, USA). In some experiments, collagen content was estimated by hydroxyproline assay, as described previously .
Lungs were inflated with 10% neutral-buffered formalin, and embedded in paraffin wax. Thin sections (5 μm) of lung were then stained with either hematoxylin and eosin or Masson's trichrome. For viral immunohistochemistry, frozen sections were prepared from infected mice, and γHV68 infection was detected using a rabbit polyclonal anti-γHV68 sera  (kindly provided by Dr Skip Virgin, Washington University School of Medicine, St. Louis, MO, USA) and detected with a goat anti-rabbit secondary antibody conjugated to alkaline phosphatase (Vectastain-ABC-AP kit for rabbit IgG (AK5001), Vector Laboratories, Burlingame, CA, USA).
TLR-9 immunohistochemistry was performed on wax-embedded tissues. The tissues were dewaxed and then blocked with H2O2 for antigen retrieval. They were incubated overnight at 4°C with a 1:50 of the primary antibody, a rabbit polyclonal antibody to TLR-9 (IMG-431; Imgenex Corp., San Diego, CA, USA). They were then incubated with biotin-labelled anti-rabbit IgG 1:500 (V1011; Vector Laboratories Inc., Burlingame, CA, USA) for 30 minutes at room temperature, followed by diaminobenzidine (catalog number 54-10-00; KPL Inc., Gaithersburg, MD, USA) for 10 minutes at room temperature. Control slides were stained with secondary antibody only.
TLR-9 immunofluorescence staining was performed on isolated cells using a mouse monoclonal primary antibody at 1:50 dilution (IMG-305A; Imgenex Corp) and Alexa Flour 555 goat anti-mouse IgG (catalog number A21424; Invitrogen Corp., Carlsbad, CA, USA). The slides were then mounted using anti-fade reagent (Prolong Gold; Invitrogen) with 4',6-diamidino-2-phenylindole (catalog number P36931, Invitrogen). Control slides were stained with secondary antibody only.
Cytokine levels were measured in whole-lung homogenates using commercial kits (R&D Systems; Minneapolis, MN, USA) according to the manufacturer's instructions. IFN-β was also measured using a commercial kit (PBL Biomedical Laboratories, Piscataway, NJ, USA).
For fibrocyte enumeration, cells obtained by collagenase digestion were incubated for 15 minutes on ice with Fc block (clone 24G2; BD PharMingen, San Diego, CA, USA) before surface staining with CD45-PerCPCy5.5 (BD PharMingen) followed by fixation and permeabilization using a kit (Cytofix/cytoperm Kit; BD PharMingen) according to the manufacturer's instructions. Cells were then blocked with goat IgG before staining with collagen 1 (rabbit anti-mouse) (Rockland, Gilbertsville, PA, USA) followed by a goat anti-rabbit phycoerythrin-conjugated secondary antibody (Jackson Immunoresearch, West Grove, PA, USA). Rabbit IgG (Jackson Immunoresearch) was used as an isotype control in place of the anti-collagen antibody. Cells were analyzed on a flow cytometer (FACSCalibur; BD Biosciences, Mountain View, CA, USA).
For other leukocyte subsets, cells were stained with Fc block followed by surface staining for CD45, CD4, CD8, CD19, Gr-1 or DX-5 using directly conjugated antibodies (BD PharMingen).
Isolation of alveolar epithelial cells
Type II alveolar epithelial cells were isolated and purified using a protocol published previously .
Isolation of fibroblasts and fibrocytes
Mice were perfused with saline, and lung lobes were removed and minced with scissors. The minced tissue was cultured for 14 days in complete media to obtain a population of plastic-adherent mesenchymal cells. Fibroblasts were then negatively selected using anti-CD45-conjugated magnetic beads. Fibrocytes were the positively selected CD45 fraction.
Isolation of dendritic cells
Bone marrow was cultured in complete media containing granulocyte-macrophage colony-stimulating factor (10 ng/ml) for 5 days. These cells were used as a positive control for TLR-9 expression by real-time RT-PCR.
real-time reverse transcriptase PCR
Primer and Probe sequences for real-time RT-PCR
AECs were fixed and stained, according to manufacturer's instructions, with an antibody (M30 Cytodeath; Roche, Indianapolis, IN, USA) that detects a caspase cleavage product of cytokeratin 18 in epithelial cells.
Caspase 3 western blotting
Western blots were performed as described previously using the follwoing antibodies: for cleaved caspase 3, a rabbit anti-human/mouse antibody (MAB835; R&D Systems) and for β-actin, a mouse monoclonal antibody (Sigma, cat# AC-74), with secondary antibodies for cleaved caspase 3 goat anti-rabbit (catalog number 31462; Pierce Protein Research Products, Thermo Fischer Scientific, Rockford, IL) and β-actin goat anti-mouse (catalog number 31432; Pierce Protein Research Products, Thermo Fischer Scientific, Rockford, IL).
Fibroblasts were seeded at 5000 cells/well in 96-well flat-bottomed plates. 3H-thymidine (1 μCi) was added to each well during the final 16 hours. Cells were harvested onto glass-fiber filters, and the incorporated radioactivity was determined by scintillation counting. In some experiments, IFN-β (Cellsciences, Canton, MA, USA) was added during culture.
In vivo CpG delivery
Specific-pathogen-free male C57BL/6 (wild-type (WT)) mice (Taconic, Germantown, NY, USA) 6 to 8 weeks of age were used. WT mice received 0.05 U of bleomycin sulfate (Blenoxane; Bristol-Meyers Pharmaceuticals, Evansville, IN, USA) dissolved in PBS (approximately 1.7 U/kg) via IT injection. Fourteen days later, all groups of mice were mildly anesthetized, and received a single bolus (50 μg) of CpG ODN (dissolved in sterile saline) by intranasal delivery. Groups of WT mice (n = 5 to 10 per timepoint) were monitored for survival. Mice were killed by cervical dislocation 28 days after the IT challenge with bleomycin, and whole-lung tissue was dissected for histological and biochemical analysis of hydroxyproline content. Untreated mice (n = 5) did not receive bleomycin, and this time point was designated as day 0.
All calculations were performed using Prism 5.0 software (GraphPad Software, San Diego, CA, USA). Values are expressed as means ± SEM. Two-sample t-tests were used for comparisons of the means when two groups were compared. One-way ANOVA was used for comparisons of three or more groups with Bonferroni or Dunnet post-hoc test analyses depending on whether all groups were compared with each other or all groups were compared with a single control group. P ≤ 0.05 was considered significant.
List of abbreviations
enzyme-linked immunosorbent assay
This work was supported by NIH grants HL087846 (BBM and GBT), AI065543 (BBM) and KO8 awards to both TRL (HL94666) and UB (HL094762).
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