Type I Interferon Signaling Contributes to Chronic ...

TOXICOLOGICAL SCIENCES 116(2), 682–692 (2010) doi:10.1093/toxsci/kfq158 Advance Access publication May 31, 2010

Type I Interferon Signaling Contributes to Chronic Inflammation in a Murine Model of Silicosis Giulia Giordano,* Sybille van den Bruˆle,* Sandra Lo Re,* Perrine Triqueneaux,* Francine Uwambayinema,* Yousof Yakoub,* Isabelle Couillin,† Bernhard Ryffel,† Thomas Michiels,‡ Jean-Christophe Renauld,‡,§ Dominique Lison,* and Francxois Huaux*,1

1

To whom correspondence should be addressed at Louvain Centre for Toxicology and Applied Pharmacology (LTAP), Universite´ catholique de Louvain, Avenue E. Mounier 53.02, 1200 Brussels, Belgium. Fax: þ32-2-764-53-38. E-mail: [email protected]. Received January 20, 2010; accepted May 20, 2010

Lung disorders induced by inhaled inorganic particles such as crystalline silica are characterized by chronic inflammation and pulmonary fibrosis. Here, we demonstrate the importance of type I interferon (IFN) in the development of crystalline silica–induced lung inflammation in mice, revealing that viruses and inorganic particles share similar signaling pathways. We found that instillation of silica is followed by the upregulation of IFN-b and IRF-7 and that granulocytes (GR11) and macrophages/ dendritic cells (CD11c1) are major producers of type I IFN in response to silica. Two months after silica administration, both IFNAR- and IRF-7–deficient mice produced significantly less pulmonary inflammation and chemokines (KC and CCL2) than competent mice but developed similar lung fibrosis. Our data indicate that type I IFN contributes to the chronic lung inflammation that accompanies silica exposure in mice. Type I IFN is, however, dispensable in the development of silica-induced acute lung inflammation and pulmonary fibrosis. Key Words: type I interferon; lung; chronic inflammation; silica; granulocytes.

Inhalation of crystalline silica particles is implicated in a variety of pulmonary disorders, including alveolitis, pulmonary fibrosis, and lung cancer. The pulmonary response induced by silica involves the marked recruitment of inflammatory cells, such as neutrophils and macrophages; the production of proinflammatory mediators, such as cytokines and eicosanoids; apoptosis; as well as the release of growth factors for fibroblasts that may collectively lead to the development of fibrosing nodules, the impairment of pulmonary function (silicosis), and possibly carcinogenesis (Hamilton et al., 2008; Huaux, 2007; Johnston et al., 2000; Porter et al., 2006; Thibodeau et al., 2004; Warheit et al., 2007). The role of the innate immune system in silicosis has been previously addressed by exploring the contribution of pattern

recognition receptors expressed by macrophages and dendritic cells (Beamer and Holian, 2007, 2008; Huaux, 2007). IL-1b is a key cytokine of the innate immune system because the cytoplasmic domain of type I IL-1 receptor is highly homologous to cytoplasmic domain of Toll-like receptors. Recent studies demonstrated that the uptake of silica particles by macrophages triggers the production of IL-1-b by activation of the ASC- and NALP-3-dependent inflammasome, which leads to neutrophil influx and activation (Hornung et al., 2008). Evidence suggests that the binding of silica particles to scavenger receptors, predominantly SR-A I/II and MARCO, is crucial for particle clearance, apoptosis, release of inflammatory mediators, and lung inflammation (Hamilton et al., 2006; Thakur, Beamer, et al., 2009; Thakur, Hamilton, et al., 2009). Type I interferon (IFN), another family of molecules belonging to the innate immune system, is of particular interest to this investigation. Type I IFN appears to be dependent of SR-A I/II and MARCO activation, and recent reports support mutual interaction between type I IFN and scavenger receptors (Murao et al., 2008; Seimon et al., 2006). The potential role of type I IFN in lung responses to silica has not been explored yet. Type I IFN comprises IFN-b, IFN-a, and many other subtypes, including newly discovered IFNs that are not well characterized. IFN-b is generally described as the fibroblast IFN, but it is also secreted in large amounts by plasmacytoid dendritic cells in the early and late phases of antiviral response (Decker et al., 2005; Stark et al., 1998; Zhang and Pagano, 2002). Cellular responses to type I IFN are mediated by interaction with a high-affinity cell-surface receptor (IFNAR). OAS (oligoadenylate synthetases) and RIG-I (retinoic acid– inducible gene), two of the many IFN-stimulated genes (ISGs) that have been identified, are pivotal in mediating the effects of type I IFN, and their expression represents a specific signature of type I IFN biological activities (Boxel-Dezaire et al., 2006;

Ó The Author 2010. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: [email protected]

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*Louvain Centre for Toxicology and Applied Pharmacology, Universite´ catholique de Louvain, 1200 Brussels, Belgium; †IEM-UMR6218, University of Orleans and CNRS, 45071 Orleans, France; ‡de Duve Institute, Universite´ catholique de Louvain, 1200 Brussels, Belgium; and §Ludwig Institute for Cancer Research, 1200 Brussels branch, Belgium

SILICA-INDUCED TYPE I INTERFERON SIGNALING

MATERIALS AND METHODS Animals. C57BL/6 mice were purchased from Charles River Laboratory (Brussels, Belgium). IFNAR-competent and -deficient mice (in 129SV and C57BL/6 backgrounds) were obtained from Guy Warnier, Ludwig Institute for Cancer Research (Brussels, Belgium), and the Transge´nose Institute (Orleans, France). IRF-7–deficient mice (C57BL/6 background) were purchased from Riken BRC (Ibaraki, Japan) with the permission of T. Taniguchi. All experiments were conducted with female mice. Animals were maintained in sterile microisolators with sterile rodent feed and acidified water and housed in positive-pressure airconditioned units (25°C, 50% relative humidity) on a 12-h light/dark cycle. The experimental protocol was approved by the local committee for animal research at the Faculty of Medicine of the Universite´ catholique de Louvain. Animal treatment. Crystalline silica particles (DQ12; d50 ¼ 2.2 lm) were obtained from Dr L. Armbruster (Essen, Germany). The particles were heated at 200°C for 2 h just prior to suspension and administration in order to sterilize the silica and inactivate any trace endotoxin. A suspension of silica in sterile 0.9% saline was introduced directly into the lungs by pharyngeal instillation (2.5 mg silica, suspended in 60 ll of saline, per mouse). This dose is previously described in the literature as effectively inducing chronic lung inflammation and fibrosis (Huaux et al., 2002). All instillations were performed on mice anesthetized with a mix of 1 mg Ketalar (N.V. Warner-Lambert, Zaventem, Belgium) and 0.2 mg Rompun (Bayer A6, Leverkussen, Germany). Bronchoalveolar lavage and whole-lung homogenates. For this study, two parallel treatment groups were used; one set of mice was reserved for collecting lavage fluid, the other for providing whole-lung homogenates. The mice in both groups were sacrificed by ip injection of 20 mg sodium pentobarbital (Certa, Braine-l’Alleud, Belgium) at selected time intervals (3, 30, and 60 days) after silica treatment. Bronchoalveolar lavage (BAL) was performed by cannulating the trachea and infusing the lungs four times with 1.5 ml of 0.9 % saline. The BAL fluid (BALF) was centrifuged (280 3 g, 4°C, 10 min) and the cell-free supernatant was used for biochemical measurements. The cell pellets of the BAL fractions were resuspended in saline to determine cell numbers and differentials on cytocentrifuge preparations stained with Diff-Quik (Baxter, Lessines, Belgium). Unlavaged lungs were perfused via the

right heart ventricle and excised. The right lobe was placed into a Falcon tube chilled on ice and 2 ml of 0.9% saline was added. The left lobe was placed into a Falcon tube with 2 ml of Trizol (Invitrogen, Merelbeke, Belgium). The lungs were then homogenized with an Ultra-Turrax T25 homogenizer (Janke and Kunkel, Brussels, Belgium) for 30 s and stored at 80°C for later use. Biochemical analyses. Lactate dehydrogenase (LDH) activity in BALF was assayed spectrophotometrically by monitoring the reduction of nicotinamide adenine dinucleotide (NADþ) at 340 nm in the presence of lactate. Total proteins in BALF were determined by the pyrogallol red staining method (Technicon RA system; Bayer Diagnostics, Domont, France). Tissue myeloperoxidase (MPO) activity was estimated in lung homogenates as previously described (Gasse et al., 2009). Hydroxyproline measurements and histology. Hydroxyproline lung content was measured by high performance liquid chromatography after hydrolysis of the lung tissue in 6 N HCl as reported previously (Huaux et al., 2002). Unlavaged whole lungs were collected and inflated with 3.6% buffered formaldehyde (Sigma, St Louis). After overnight fixation, they were embedded in paraffin, 5-lm thick sections were taken, and stained with eosin-hematoxylin or Masson’s Trichrome. RNA extraction and quantification. Total RNA was isolated from the lung homogenates in Trizol by following the manufacturer’s protocol. The samples were treated with DNase (Ambion, Lennik, Belgium) and RNA was reverse transcribed using Superscript III Reverse Transcriptase (RT; Invitrogen). The resulting complementary DNA was then diluted 10- or 50-fold in sterile nanopure water and used as a template in subsequent real-time polymerase chain reactions (PCR). Sequences of interest were amplified with the following forward: 5#-AGAGG GAAATCGTGCGTGAC-3# (b-actin); 5#-GGCAGACCCCGTCCCA-3# (IRF7); 5#-ATGAACAACAGGTGGATCCTCC-3# (IFNb); 5#-GGATGCCTGGGAGAGAATCG-3# (OAS-l2); and 5#-AAGATTCTGGACCCCACCTACA -3# (RIG-I); and reverse primers: 5#-CAATAGTGATGACCTGGCCGT-3# (b-actin); 5#-AAGGCTGCGCTCGGTG-3# (IRF-7); 5#-AGGAGCTCCTGACATTTCCGAA-3# (IFNb); 5#-TCGCCTGCTCTTCGAAAC TG-3# (OAS-l2); and 5#ATTGGGCCCTTGTTGTTCTTC-3# (RIG-I) (Invitrogen). Here, b-actin served as a reference gene. Quantitative PCR was performed on an ABI 7000 (Applied Biosystems, Foster City) under the following conditions: 50°C 2 min, 95°C 10 min, (95°C 15 s, 60°C 1 min) 3 40 cycles. Standards and samples (5 ll) were amplified using SYBR green PCR Master Mix (Applied Biosystems, Lennik, Belgium) in a total volume of 25 ll, and the degree of gene expression was given as a ratio (gene expression per sample to gene expression of the reference gene). Some of the RNA samples were treated with the reverse transcriptase kit without enzyme to ensure there was no genomic DNA contamination (especially important for IFN-b, which is coded by a sequence without intron). GR1þ and CD11cþ cell purification. Whole lungs from control and silicatreated mice were mechanically digested and incubated with 2.5 mg Liberase CI/lung (Roche Diagnostics, Brussels, Belgium) and 0.25 mg DNase/lung (Worthington, Brussels, Belgium). Granulocytes (GR1þ) were purified from the digested tissue on MACS Separation Columns (LS Columns Miltenyi Biotec, Utrecht, The Netherlands) using anti-mouse GR1 antibodies coupled with magnetic microbeads (BD Pharmingen, Aalst, Belgium). GR1-positive cells were additionally purified by flow cytometry cell sorting (FACSVantage; BD Biosciences) based on their size (Forward Scatter) and granularity (Side Scatter). Macrophages/dendritic cells (CD11cþ) were purified from MACS GR1-depleted flow-through fraction by using anti-CD11c antibodies with MACS microbeads and MACS columns. The negative fraction of this magnetic separation represents the CD11c/GR1 population. The purified GR1þ and CD11cþ preparations were assessed on cytocentrifuge slides stained with Diff-Quick and showed 95% purity. Purified cells were counted, resuspended in dulbecco’s modified eagle medium (DMEM)-fetal bovine serum (FBS) 10% (106 cells/ml), and cultured (100 ll of cell suspension per well) for 12 h in 96-well plates. Supernatants were then collected and centrifuged prior to IFN-b quantification by bioassay. ELISA and bioassay. IFN-b and IFN-a were quantified by ELISA (detection limits ¼ 7 and 6 pg/ml, respectively; PBL Biomedical Laboratories,


Seth et al., 2006; Theofilopoulos et al., 2005). The family of transcription factors IFN regulatory factor (IRF) is also well known to play a fundamental role in the induction of and response to type I IFN. In the 10 members of the mammalian IRF family (IRF-1 to IRF-10) (Honda and Taniguchi, 2006; Honda et al., 2005; Taniguchi et al., 2001), IRF-7 is weakly expressed in most cell types and is strongly induced by type I IFN-mediated signaling but, at the same time, is the major inducer of type I IFN production which, in turn, allows for vigorous production of type I IFN (Honda and Taniguchi, 2006; Zhang and Pagano, 2002). The upregulation of IRF-7 is, therefore, both a consequence and a cause of type I IFN expression (Sato et al., 1998). Our preliminary data suggested that components of antiviral pathways may also be involved in the lung reaction to silica; therefore, we decided to test the hypothesis that type I IFN plays a significant role in this reaction. We assessed the expression of type I IFN in the lungs of 129SV and C57BL/6 mice, examined pulmonary responses of IFNAR-competent and -deficient mice and IRF-7–competent and –deficient mice, and identified the major cellular sources of type I IFN after in vivo treatment with silica.

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Piscataway, NJ) in whole-lung homogenates. Murine IL-1b, IL-6, TNF-a, KC, and CCL2 concentrations were measured in BALF by ELISA kits (R&D Systems) following the manufacturer’s protocols. Biologically active type I IFN was quantified by bioassay (Rubinstein et al., 1981) based on the capacity of type I IFN to specifically and strongly induce OAS-l2 gene expression in L929 cells. L929 cells were resuspended in DMEM þ 10% FBS at a density of 3.5 3 105/ml, plated with 100 ll cell suspension/well in 96-well plates, and incubated overnight at 37°C with 5% CO2 in a humidified incubator. Medium alone (negative control), mouse recombinant IFN-b (positive control, 10 U/ml, PBL Biomedical Laboratories), and lung purified cell culture supernatants (see above) were incubated for an additional 24 h. Medium was then aspirated from the wells, messenger RNA was isolated in Trizol, and OAS-l2 gene expression was assessed by qRT-PCR as detailed above.

RESULTS

Signature of Type I IFN-Related Responses in the Lungs of Silica-Treated Mice First, we performed a macroarray analysis targeting genes linked with the innate immune system (SuperArray Bioscience, Boechout, Belgium) on pulmonary samples obtained from silica-treated 129SV or C57BL/6 mice sacrificed at different time points (days 3, 30, and 60). This preliminary analysis showed that several genes involved in antiviral pathways were upregulated following silica exposure (not shown). Among these genes, IRF-7, a key transcription factor controlling IFN-b expression and activity (Sato et al., 1998), was strongly upregulated after silica treatment. To confirm this observation, we used qRT-PCR and ELISA to assess the expression of IRF-7 and IFN-b in whole lungs at 3, 30, and 60 days after instillation of silica in 129SV mice. We found that both IRF-7 (Fig. 1A) and IFN-b (Fig. 1B) gene expression were significantly elevated by silica at the longer time points (days 30–60). The levels of IFN-b in lung homogenates as measured by ELISA also increased significantly at 60 days in silicaversus saline-treated animals (Fig. 1C). IFN-a expression was not modified by silica exposure (not shown). These observations suggest that type I IFN expression is induced by silica during chronic silicosis in 129SV mice. Lung Granulocytes and Macrophages/Dendritic Cells are Major Sources of Type I IFN in Response to Silica The lung inflammatory response to silica is characterized by a marked influx of neutrophils and macrophages, both of which are prominent cellular sources of inflammatory and fibrotic mediators (Huaux, 2007). To determine whether these inflammatory cells are capable of producing type I IFN after silica administration, we purified lung GR1þ (granulocytes) and CD11cþ (macrophages and dendritic cells) cells from saline- and silica-treated 129SV mice and assayed for IFN-b expression by ELISA at day 60, when IFN-b expression is known to be high (Fig. 1C).

FIG. 1. Silica particles induce lung expression of IRF-7 and IFN-b in 129SV mice. The expression of IRF-7 (A) and IFN-b (B) was assessed by qRT-PCR in whole-lung homogenates of 129SV mice treated with sterile saline (0.9% NaCl) or sterile crystalline silica particles suspended in saline (2.5 mg/ 60 ll) at 3-, 30-, and 60-day time points. The expression of IFN-b and IRF-7 were normalized to b-actin. IFN-b levels (C) in whole-lung homogenate were estimated by ELISA in both saline- and silica-treated groups. Each bar represents the mean ± SEM of three to five observations. *p < 0.05, **p < 0.01 (Mann-Whitney test relative to saline-treated mice); ND, not detected.

IFN-b levels were undetectable by ELISA in both freshly isolated lung cells and in cultured cells. We switched to using bioassay because it is well recognized that the L929 fibroblast test is sensitive and allows for the quantification of type I IFN bioactivity. This assay is based on the capacity of type I IFN to specifically induce ISG expression, such as OAS-l2 in fibroblast cultures. The purity of our cell preparations


Statistics. Differences between the experimental groups were evaluated with one-sided unpaired Student t-test or Mann-Whitney tests as appropriate. Statistical significance was considered at p < 0.05.


The Induction of Type I IFN in Silica-Treated Mice Is Enhanced by Type I IFN Itself To examine whether upregulation of IFN-b and IRF-7 is the consequence of increased IFN-b production in the lungs of silica-treated mice, we treated type I IFN-receptor (IFNAR)deficient mice and their wild-type congeners (129SV) with silica and assessed the lung expression of IRF-7 (Fig. 3A) and IFN-b

(Fig. 3B) at day 60. qRT-PCR results clearly demonstrated that in absence of IFNAR, silica exposure failed to induce an upregulation of IRF-7 and IFN-b. Therefore, we concluded that the increased lung expression of IRF-7 and IFN-b induced after silica treatment is enhanced by type I IFN. Using IFNARdeficient mice allowed us the opportunity to further investigate the role of IFN-b and IRF-7 in experimental silicosis. Role of the Type I IFN Pathway in Silica-Treated Mice To determine whether type I IFN is implicated in the development of lung inflammation, injury, and fibrosis induced by silica, we investigated these responses to silica in 129SV IFNAR-deficient mice. The results show that 2 months after silica treatment, 129SV IFNAR/ mice developed a reduced accumulation of inflammatory cells in the lung in comparison with wild-type mice (Fig. 4A–E). Our histological studies confirm these observations (Fig. 5A–B) and show fewer inflammatory cells in the alveoli of IFNAR mice compared with 129SV animals. Tissue injury induced by silica was estimated by the measurement of LDH activity as well as total

FIG. 2. The administration of silica upregulates type I IFN in GR1þ and CD11cþ lung cells. CD11cþ (macrophages/dendritic cells) and GR1þ (granulocytes) lung cells were harvested from saline- and silica-treated 129SV mice at the 60-day time point and purified by MACS and FACS. Purified cell populations from the silica-treated group are illustrated (Diff-Quick staining) (A and B). Original magnifications 3400. (C) The release of biologically active type I IFN by purified CD11cþ, GR1þ, and CD11c/GR1 lung cells from both saline- and silica-treated groups was assessed by L929 bioassay. Medium alone (m) and mouse recombinant IFN-b (rIFN-b) were used as negative and positive controls, respectively. Each bar represents the mean ± SEM of three observations. Exposure to rIFN-b and CD11cþ/GR1þ conditioned media resulted in significant increases in OAS-l2 expression compared with exposure to medium alone or to CD11cGR1 cell culture supernatants (see ‘‘Materials and Methods’’ section).


was > 95% as assessed by Diff-Quick staining (Fig. 2A–B). We observed that purified GR1þ and CD11cþ cells were major sources of type I IFN because culture supernatants of these cells were able to induce OAS-l2 expression in L929 cells. CD11c/GR1 cells were, in contrast, inactive (Fig. 2C). This effect was particularly marked in cells from silica-treated mice. IRF-7 as well as RIG-I gene expression were also upregulated after silica treatment in purified GR1þ and CD11cþ cells (data not shown). Together, these observations support the hypothesis that pulmonary granulocytes and macrophages/dendritic cells release biologically active type I IFN after silica treatment.

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release of chemokines (KC, Fig. 6A, and CCL2/MCP-1, Fig. 6B) is abrogated in deficient animals compared with their competent counterparts. In contrast, IL-1b (Fig. 6C), IL-6, and TNF-a (not shown) levels were not modified in treated IFNAR animals. We concluded that type I IFN regulates lung chemokine expression during the late phases of silica-induced lung inflammation.

proteins in BALF. LDH (Fig. 4F) and protein (Fig. 4G) levels were significantly lower in silica-treated IFNAR/ mice compared with wild-type mice. No difference was observed between IFNAR-deficient and 129SV mice during the short-term lung responses induced by silica (day 3, cellular inflammation and tissue injury, data not shown). In addition, type I IFN did not seem to play a role in the fibrogenic response to silica (60 days) because neither collagen quantitation (Fig. 4H) nor histological analysis (Fig. 5C–D) revealed significant differences in the fibrotic response between IFNAR-deficient and wild-type mice. Taken together, these results imply that type I IFN contributes to the chronic inflammatory response induced by silica but may be dispensable in the development of acute lung inflammation and pulmonary fibrosis. Type I IFN Mediates Chemokine Expression during the LongTerm Lung Responses Induced by Silica We conducted further studies to clarify whether type I IFN participates in chronic lung inflammation through the release of cytokines and/or chemokines implicated in experimental silicosis. At day 60, we observed that silica-induced marked

DISCUSSION

In both humans and experimental animals, inhalation of silica causes a chronic inflammatory lung reaction (alveolitis), which may lead to the development of lung fibrosis and/or cancer. In the last decade, several studies have helped clarify the mechanisms of silica-induced alveolitis (Huaux, 2007). The inflammatory pathway classically described includes the uptake of silica by macrophages through an interaction with receptors of the innate immune system (Brown et al., 2006; Hamilton et al., 2006). Following silica uptake, alveolar macrophages are activated and consequently release a cocktail of proinflammatory cytokines, such as TNF-a, IL-6, and IL-1b. These activate lung epithelial cells to release chemokines, such as MIP-2 and KC, which then contribute to the recruitment of inflammatory


FIG. 3. The induction of IRF-7 and IFN-b in the lungs of silica-treated mice is mediated by the type I IFN receptor (IFNAR). The expression of IRF-7 (A) and IFN-b (B) in whole lungs of 129SV- and IFNAR-deficient mice 60 days after silica or saline treatment was assessed by qRT-PCR. Each bar represents the mean ± SEM of three to eight observations. ***p < 0.001 (t-test relative to silica-treated competent mice).

Type I IFN Contributes to Chronic Alveolitis in the Lungs of Mice Treated with Silica Additionally, we studied type I IFN–related gene expression in C57BL/6 mice and found that in this particular strain, IRF-7 (Fig. 7A) and IFN-b (Fig. 7B) were significantly induced by silica during both the short-term (d3) and the long-term (d30 and d60) lung responses. Both early and late upregulation in C57BL/6 mice was confirmed by ELISA measurements, which showed increased IFN-b content in the lungs of silicatreated C57BL/6 mice at each time point (saline ¼ 12 ± 4, silica-d3 ¼ 27 ± 6, silica-d30 ¼ 41 ± 8, and silica-d60 ¼ 31 ± 5). The C57BL/6 model allowed us to hypothesize that type I IFN plays an important role in acute as well as chronic alveolitis. We therefore compared cellular inflammation and lung tissue injury at 3 and 60 days after silica treatment in C57BL/6 IFNAR- and IRF-7–deficient mice. Although the early (day 3) lung accumulation of neutrophils (BAL numbers, Fig. 7C and MPO activity, not shown), macrophages, and lymphocytes (not shown) as well as early lung injury (LDH levels, Fig. 7E; protein levels, not shown) were similar in C57BL/6, IFNAR/, and IRF-7/ mice, late lung inflammation (day 60) was significantly reduced in the deficient animals exposed to silica in comparison with their wild-type counterparts (Fig. 7D and 7F). Lung fibrosis analysis by OH-proline revealed that collagen content was not modified by IFNAR or IRF-7 deficiencies (Fig. 7G). These data confirm that in mice, type I IFN contributes to the maintenance of chronic lung inflammation during silicosis.


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Downloaded from http://toxsci.oxfordjournals.org at University of Michigan on August 3, 2010 FIG. 4. Type I IFN contributes to the chronic lung inflammatory response to silica in 129SV mice. The total number of cells (A), macrophages (B), neutrophils (C), and lymphocytes (E) was counted in the BALF of 129SV and IFNAR/ mice 60 days after the administration of silica or saline. BAL LDH (F) and protein (G) levels as well as lung tissue MPO (D) and OH-proline (H) contents from saline- or silica-treated 129SV- and IFNAR-deficient mice are shown at day 60. Each bar represents the mean ± SEM of three to seven observations. *p < 0.05 (t-test relative to silica-treated competent mice).

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cells (Fubini and Hubbard, 2003; Hornung et al., 2008; Yuen et al., 1996). In this study, we observed reduction of the long-term inflammatory response in IFNAR- and IRF-7–deficient mice that were treated with silica compared with their competent counterparts. We found that IFN-b, which is a member of type I IFN (Malmgaard, 2004; Stetson and Medzhitov, 2006; Vilcek, 2006) and originally characterized as an antiviral mediator, was also upregulated in our silica model. Both viruses and inorganic particles, therefore, appear to share a similar signaling pathway, although the degree of expression of IFN-b and its related genes such as IRF-7 in response to silica is much lower than during a viral infection (Delhaye et al., 2006; Honda et al., 2005). However, these lower levels are sufficient enough to maintain chronic inflammation because the long-term effects of silica exposure in IFNAR- and IRF-7–deficient mice are characterized by reduced inflammatory reaction. The mechanisms by which type I IFN stimulates recruitment of immune cells in inflamed mouse lung are not clearly understood. Type I IFN can trigger the production of chemokines capable of inducing inflammatory chemotaxis, such as CXCL10, MIP-1, CCL2/MCP-1, and RANTES (McManus et al., 2000; Zhou et al., 2007). We demonstrated that KC and

CCL2, two major inflammatory chemoattractants for neutrophils and macrophages recruitment, respectively (Bless et al., 2000; Michalec et al., 2002), and strongly induced by silica in mice (Pryhuber et al., 2003), are abrogated in absence of type I IFN. In contrast, the master proinflammatory cytokines IL-1b, TNF-a, and IL-6 were not modulated by type I IFN. We also found that the short-term reaction to silica appears independent or less dependent on type I IFN signaling, suggesting that several other major proinflammatory pathways activated during early response to silica (i.e., IL-1b, TNF-a, and IL-6) may be preponderant at this early stage and probably mask the influence of the type I IFN pathway. We conclude that lung expression of IFN-b in silica-treated mice is driven by macrophages/dendritic cells as well as pulmonary granulocytes that are recruited during inflammatory reactions to silica. In other diseases in which type I IFN are implicated (Baccala et al., 2007; Banchereau and Pascual, 2006), dendritic cells and macrophages are the major type I IFN producers. Here, for the first time, we have documented that pulmonary granulocytes also produce type I IFN and that pulmonary granulocytes are involved in the type I IFN response (pulmonary granulocytes overexpress IRF-7, OAS-l2 and RIG-I). Furthermore, we provide strong evidence that a new pathway is involved in chronic alveolitis, one in which


FIG. 5. Reduced chronic alveolitis but similar fibrotic lung lesions in IFNAR-deficient mice. Representative lung sections stained with H&E (A and B) and Masson Trichrome (C and D) from 129SV-deficient (A and C) and IFNAR-deficient mice (B and D) treated with silica at day 60. The ‘‘alveoli’’ of deficient mice contained fewer ‘‘inflammatory cells’’ compared with their competent counterparts. The prevalence of silicotic nodules and pulmonary fibrosis (in green) were similar in both strains. Original magnification was 3400.


granulocytes enhance the recruitment of additional inflammatory cells into the lung through the production of type I IFN, thereby contributing to the maintenance of chronic inflammation. We reveal that not only macrophages and epithelial cells initiate the inflammatory reaction to silica through the release of cytokines and chemokines but that granulocytes also play a significant role in the recruitment process by releasing low levels of type I IFN. Several studies underlined

that the carcinogenic activity of silica can be attributed to chronic inflammation and activated neutrophils (Knaapen et al., 2006). Our study newly identifies type I IFN as important cytokines specifically implicated in chronic alveolitis. These cytokines may thus represent new targets for reducing the impact of persistent lung inflammation in silicosis. We know that human patients and laboratory rats developing silicosis have persistent alveolitis, which led to the original hypothesis that protracted inflammation is a cause of lung fibrosis (Rimal et al., 2005). However, recent experimental studies have challenged the paradigm that pulmonary inflammation drives lung fibrosis and have proposed that, at least in some cases, the pathological process is independent of inflammation and is instead mediated by alternative mechanisms involving fibrocytes, coagulation cascades, proteases, epithelial-mesenchymal transition, and immunosuppressive immunity (Hardie et al., 2009; Huaux, 2007; Strieter and Mehrad, 2009). Several studies using silica-treated mice (Adamson et al., 1992, 1994; Beamer and Holian, 2005; Huaux et al., 1998; Thakur, Beamer, et al., 2009) report that lung fibrosis is indeed disconnected from inflammation, which strongly supports the hypothesis that pulmonary inflammation in the silica model does not necessarily drive fibrotic disease. In human, it is noteworthy that corticosteroids have, at best, limited efficacy in the treatment of silicosis (Greenberg et al., 2007). The results of our study clearly demonstrate that the reduction of chronic inflammation in type I IFN–deficient mice is not accompanied by a concomitant reduction in lung fibrosis, corroborating the previous findings that fibrosis induced by silica is uncoupled from inflammation, under some conditions. It remains to determine exactly how silica exposure activates IFN-b and IRF-7 expression. It is possible that in granulocytes, the mechanism of type I IFN production is similar to that of macrophages, where the expression of type I IFN in response to viral components is mediated by a TIR-domain-containing adapter-inducing interferonb-dependent pathway activated by TLR3 stimulation (Yamamoto et al., 2003), or that the mechanism is like that of plasmacytoid dendritic cells where, following the recognition of viral DNA and RNA by TLR7/8 and 9, the activation of MyD88 is needed to induce the expression of type I IFN (Gilliet et al., 2008). Another hypothesis is that cellular debris released during apoptosis or necrosis (Hamilton et al., 2006; Otsuki et al., 2007; Pfau et al., 2002) triggers the activation of cytosolic receptors, such as RIG-I, MDA5 (melanoma differentiation–associated gene 5), or DAI (double-stranded DNA-binding protein) (Muruve et al., 2008). It is interesting to note that a similar activation mechanism has been described in other autoimmune diseases associated with silica exposure (Banchereau and Pascual, 2006), including scleroderma, rheumatoid arthritis, and systemic lupus erythematosus.


FIG. 6. Type I IFN contributes to chemokine release during the chronic lung inflammatory response to silica in 129SV mice. The levels of KC (A), CCL2/MCP-1 (B), and IL-1b (C) were measured in the BALF of 129SV and IFNAR/ mice 60 days after the administration of silica or saline. Each bar represents the mean ± SEM of three to seven observations. *p < 0.05 (t-test relative to silica-treated competent mice).

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FIG. 7. Type I IFN contributes to the chronic lung inflammatory response to silica in C57BL/6 mice. The expression of IRF-7 (A) and IFN-b (B) in wholelung homogenates of C57BL/6 mice treated with either saline or silica at 3-, 30-, and 60-day time points was assessed by qRT-PCR. The expression of IFN-b and IRF-7 was normalized to b-actin. The total number of neutrophils was counted in the BALF of C57BL/6, IFNAR/, and IRF-7/ mice 3 days (C) and 60 days (D) after administration of silica or saline. BAL LDH levels from saline- or silica-treated C57BL/6, IFNAR/, and IRF-7/ mice are shown at days 3 (E) and 60 (F). Lung tissue OH-proline levels are also shown (G) for saline- or silica-treated C57BL/6 IFNAR/, and IRF-7/ mice at day 60. Each bar represents the mean ± SEM of three to seven observations. *p < 0.05, **p < 0.01 (Mann-Whitney or t-test relative to saline-treated mice [A–B] or silica-treated competent mice [C–G]).


FUNDING

Fonds de la Recherche Scientifique Me´dicale, Actions de Recherche Concerte´es, Communaute´ Francxaise de Belgique, Direction de la Recherche Scientifique, Contract no. ARC 09/ 14-021 and by the European Commission under FP7HEALTH-F4-2008, Resolve, Contract no. 202047.

ACKNOWLEDGMENTS

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F.H. is a Research Associate with the Fonds de la Recherche Scientifique (FNRS), Belgium.

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