Chronic Silicosis Upregulates Fibrogenic and Redox-related but not Proinflammatory Genes and Proteins in a Rat Model of Chronic Silicosis R.J. Langley, N.C. Mishra, J.C. Pena-Philippides, J.C. Seagrave, S.P. Singh, M.L. Sopori Lovelace Respiratory Research Institute, Albuquerque, NM Silicosis, a fibrotic granulomatous lung disease, may occur through accidental high-dose or occupational inhalation of silica, leading to acute/accelerated and chronic silicosis, respectively. While chronic silicosis has a long asymptomatic latency, lung inflammation and apoptosis are hallmarks of acute silicosis. In animal models, histiocytic granulomas develop within days after high-dose intratracheal silica instillation. However, following chronic inhalation of occupationally relevant doses of silica, discrete granulomas resembling human silicosis arise months after the final exposure without significant lung inflammation/apoptosis. To identify molecular events associated with chronic silicosis, lung RNAs from controls or chronically silica-exposed rats were analyzed by Affymetrix at 28 wk after silica exposures. Results suggested a significant upregulation of 144 genes and downregulation of seven genes. The upregulated genes included complement cascade, chemokines/chemokine receptors, G-protein signaling components, metalloproteases, and genes associated with oxidative stress. To examine the kinetics of gene expression relevant to silicosis, qPCR, ELISA, Luminex-bead assays, Western blotting, and/or zymography were performed on lung tissues from 4 d, 28 wk, and intermediate times after chronic silica exposure and compared with 14 d acute silicosis samples. Results indicated that genes regulating fibrosis (secreted phosphoprotein-1, CCL2, and CCL7), redox enzymes (superoxide dismutase-2 and arginase-1), and the enzymatic activities of matrix metalloproteinases 2 and 9 were upregulated in acute and chronic silicosis; however, proinflammatory cytokines were strongly upregulated only in acute silicosis. Thus, inflammatory cytokines are associated with acute but not chronic silicosis; however, genes regulating fibrosis, oxidative stress, and metalloproteases may contribute to both acute and chronic silicosis. • Chronic silica inhalation upregulates the expression of profibrotic and redox genes, but surprisingly without significantly affecting the expression of typical proinflammatory genes. • It is unlikely the prototypic proinflammatory cytokines are critical in the development of chronic silicosis. • Expression of redox enzymes is increased in both acute and chronic silicosis; however, in chronic silicosis, these redox enzymes had not changed at 4 d after the end of silica exposure, but were clearly changed at 7 wk after silica treatment when granulomatous changes began. • Profibrotic expression of SPP1 and CCL2 is presented in acute and initiated early in the development of chronic silicosis. • MMP2 and MMP9 are enzymatically activated as early as 7 wk post-silica inhalation; their activity is also increased in acute silicosis. These enzymes could lead to breakdown of extracellular matrix in silicosis. • The observation that corticosteroids are ineffective in preventing the development of silicotic granulomas in humans supports the expendable role of lung inflammation in the granulomatous process in silicosis; however, Spp1 may be a potential target for therapeutic intervention in silicosis. Methods Conclusions Chronic silicosis: Lewis rats were exposed to 6.2 mg/m 3 aerosolized silica (Min-U-Sil 5; U.S. Silica, Mill Creek, OK) with an average particle size of 1.75 ± 0.05 μm (mass median aerodynamic diameter) 6 h/d, 5 d/wk (Monday–Friday) for 6 wk. Control animals received filtered air under similar inhalation conditions. Acute silicosis: Rats received a single high-dose intratracheal instillation of 35 mg silica in 750 μl of saline or saline alone (control). Sacrificed 14 d post-silica exposure. Microarray analysis: Performed at the Keck-UNM Genomics Resource (University of New Mexico Cancer Research Center, Albuquerque, NM). Affymetrix GeneChip Rat 230 2.0 arrays. Data were filtered twice, a fold change filter (2 fold up or down), and the Statistical Analysis of Microarray software (http://www-stat.stanford.edu/~tibs/SAM/ ), to find all genes with a < 10% false positive q-score. Real-time PCR (qPCR): Prism 7900 HT Sequence Detection System (ABI, Foster City, CA) using standard protocols. Primers and probes for arginase-1 (Arg1), ȕ-Actin, interferon-Ȗ (IFN-Ȗ), interleukin-1ȕ (IL-1ȕ), CCL7, matrix metalloproteinase 12 (MMP12), superoxide dismutase-2 (SOD2) and secreted phosphoprotien 1 (Spp1) were designed with Primer Express 2.0. ELISA-based assay: kits were purchased from R&D System (Minneapolis, MN) and used to quantitate Spp1 (osteopontin) and CCL2 (MCP-1) in BAL fluid according to manufacturer’s instruction. Multiplex Immunoassays: The LINCOplex Rat Cytokine/Chemokine Multiplex Immunoassay (LINCO Research Inc., St. Charles, MO) was performed per manufacturer’s instructions on BAL fluid (4 d and 28 wk post exposure) to determine protein expression of 24 cytokines and chemokines. Western blot analysis: Whole lung lysates probed for Goat antiserum SOD2, Arg1, MMP12, and Actin antibodies (Santa Cruz Biotechnologies, Santa Cruz, CA). Matrix Metalloprotease Zymography: Performed on 7.5% SDS gels with 0.15% gelatin. Incubated for 24 h at 37˚C in 100 mM Tris (pH 7.4, containing 5 mM CaCl 2 and 10 μM ZnCl 2 ). The bands were analyzed using a Bio-Rad GS-800 scanner and Quantity One software (Bio-Rad). Statistics: GraphPad Prism Software 3.0 (GraphPad Inc., San Diego, CA); p values of ≤ 0.05 were considered significant. Error bars in all figures represent the standard error of mean. Acknowledgements These studies were supported in part by grants from the NIH (R01 DA017003 and R01 DA04208-17), from the U.S. Army (W81XWH-04-C-0071), and LRRI internal funds. We thank Dr. Gavin Pickett and Mrs. Marilee Morgan of the Keck-UNM Genomics Resource (University of New Mexico Cancer Research Center, Albuquerque, NM) for their help with Affymetrix analysis. Results (Continued) Results Abstract Results (Continued) Figure 1. Il-1ȕ and IFN-Ȗ are upregulated in acute but unchanged in chronic silicosis. Changes seen in whole lung mRNA (A) IL-1ȕ and (B) IFN-Ȗ as determined by qPCR in chronic silicosis (4 d, 7 wk, 14 wk, 28 wk; low dose post-silica inhalation exposure) and acute silicosis (Acute; 14 d high dose post-silica intratracheal exposure). ** p < 0.005; *** p < 0.0001. Figure 2. Silica exposure leads to moderate increases in oxidative markers, while acute silica exposure leads to robust changes in oxidative markers. Oxidative stress markers (A) SOD2 and (B) Arg1 in chronic silica (4 d and 28 wk post-exposure) and acute silica exposures in the whole lung mRNA (qPCR) (C). Whole lung lysate Western Blot analysis of oxidative stress markers (SOD2, Arg1) over the time course of chronic and acute silica models for controls (C) and silica (SL). * p < 0.05; ** P < 0.005; *** p < 0.0001. Densitometry analysis of Western Blots for (D) SOD2 and (e) Arg1. Lanes were normalized against the density readings of ȕ-Actin and are presented as fold change as compared to controls. Table 1. Genes Displaying Significant Changes in the Rat Lungs of 28 Week Post Silica Exposure Gene Gene Name Fold Change q- score Gene Gene Name Fold Change q-score Lpo Lactoperoxidase 101.4 6 C3 complement component 3 2.51 5.16 Orm1 orosomucoid 1 19.4 8.37 Rgs1 reg. of G-protein signaling 1 2.47 9.15 Saa3 Serum A A3 14.7 7.85 Gzmm lymphocyte Met-ase 1 2.33 4.92 MMP12 matrix metalloproteinase 12 11.4 4.92 HMOX1 Heme oxygenase 1 2.33 4.46 CCL7 C-C motif chemokine 7 9.09 4.92 Nkg7 natural killer cell group 7 2.33 5.58 CCL2 C-C motif chemokine 2 8.55 3.96 Top2a topoisomerase (DNA) 2 alpha 2.31 4.62 Pla2g2a Phospholipase A2, group IIA 4.76 9.31 Slpi secretory leukocyte protease inhibitor 2.29 6.34 Mcpt2 mast cell protease 2 4.08 7.7 C2 complement component 2 2.28 2.6 Lcn2 lipocalin 2 3.75 4.92 Gpr9 G protein-coupled receptor 9 2.26 7.7 Chia chitinase, acidic 3.58 4.92 Mcpt9 mast cell protease 9 2.2 4.92 Fcgr2b Fc receptor, IgG, low affinity IIb 3.3 6.61 Fcer1a Fc receptor, IgE, HA I, alpha 2.19 3.6 Itgam integrin alpha M (aka CD11b) 3.25 5.9 Cdc2a cell division cycle 2 homolog A 2.17 5.58 Arg1 arginase 1 3.14 4.92 Zap70 zeta-chain (TCR) 70kDa 2.17 2.6 Chi3l1 chitinase 3-like 1 2.98 4.92 Pik3cd phosphoinositide-3-kinase cat. d 2.17 8.37 Hp haptoglobin 2.93 7.85 Tubb2b beta-tubulin T beta15 2.14 4.92 Cd2 CD2 antigen 2.9 7.7 SOD2 superoxide dismutase 2 2.14 8.37 Pumag Puma-g 2.87 6 Lat linker for activation of T cells 2.12 4.46 Mcpt10 mast cell protease 10 2.87 8.37 Mct3 monocarboxylate transporter 2.12 8.37 CCL3 chemokine (C-C motif) ligand 3 2.87 2.6 Dmbt1 deleted in malig. brain tumors 1 2.11 5.9 Pttg1 pituitary tumor-transforming 1 2.84 6.34 RT1-Aw2 RT1 class Ib, locus Aw2 2.09 5.58 Ctsk cathepsin K 2.83 4.92 Cyba cytochrome b558 alpha-subunit 2.07 4.92 Spp1 secreted phosphoprotein 1 2.8 3.96 Gpnmb glycoprotein nmb 2.07 7.85 C4bpa complement 4 binding prot. Α 2.75 2.6 Bcl2a1 BCL2-related protein A1 2.07 4.92 Mt1a Metallothionein 2.74 7.85 RT1-N1 RT1 class Ib, H2-TL-like, grc (N1) 2.07 7.7 Cdc20 cell cycle protein p55CDC 2.69 4.92 C2ta MHC class II transactivator 2.06 5.58 Cfi complement factor I 2.67 2.6 Lst1 leucocyte specific transcript 1 2.06 6.85 Ns5atp9 Ns5atp9 protein 2.59 5.58 Gro1 gro 2.06 6.85 Scya4 small inducible cytokine A4 2.56 4.92 Fetub fetuin beta 2.05 2.62 C1qb complement 1,q beta 2.54 6.61 Dlm1 Macrophage protein DLM-1 2.04 5.9 Cd3d CD3 antigen delta polypeptide 2.52 4.92 Tcrb T-cell receptor beta chain 2.02 3.6 Figure 3. Silica exposure increases pro-fibrotic chemokines in chronic and acute silica exposure during granuloma formation. Whole lung mRNA changes of chemokines (A) Spp1 and (B) CCL7 as determined by qPCR. (C) Spp1 and (D) CCL2 ELISA of BAL fluid (five rats per group) over the time course of chronic and acute silica exposures. ** p < 0.001; *** p < 0.0001. Table 2. Cytokines and Chemokines were Upregulated in Acute but not Chronic Silica Exposure Cytokine/ Chemokine Control (pg/ml) 4-d exposure (pg/ml) 28-wk exposure (pg/ml) Acute exposure (pg/ml) GM-CSF 14.6 ± 3.9 6.1 ± 1.3 6.6 ± 1.0 31.4 ± 4.4 * CCL2 5.9 ± 1.3 5.530 ± 1.1 13 ± 3.4 226 ± 25 *** Leptin 2.1 ± 0.6 1.4 ± 0.4 1.0 ± 0.1 63 ± 32 * MIP-1α BD BD BD 6.9 ± 2.4 †# IL-1ȕ 1.5 ± 0.3 BD BD 12.8 ± 4.1 ** IL-6 27.5 ± 5.3 27.3 ± 15 28 ± 5 141.2 ± 49 ** IL-9 52 ± 34.4 5.4 ± 2.7 9.3 ± 4.3 1960 ± 924 * IL-13 2.6 ± 1.0 BD 2.6 ± 2.3 10.6 ± 8.6 IL-10 2.8 ± 0.6 2.2 ± 0.7 19.4 ± 17 2697 ± 2568 IL-12(p70) 2.2 ± 0.5 1.7 ± 0.9 1.6 ± 0.51 4.9 ± 0.7 * IFN-Ȗ 1.8 ± 0.5 BD BD 314 ± 144 ** IL-17 1.9 ± 0.3 BD 2.2 ± 0.5 50.4 ± 24 ** IL-18 37.4 ± 15 5.2 ± 2.3 85 ± 58.4 4348 ± 1743 ** Gro-Kc 18.4 ± 5.3 15.0 ± 2.3 15 ± 3.4 204.1 ± 4.2 *** Rantes 148.0 ± 18.1 130.4 ± 18.1 149.2 ± 20.2 270.5 ± 10 ** TNF-α BD BD BD 2.8 ± 0.5 †# BD: Below detection limits †#: Unable to perform reliable statistics. Tested, but no changes seen or below detection limits: Eotaxin, G-CSF, IL-1α, IL-2, IL-5, IP-10 Figure 4. While MMP12 protein level is unchanged, MMP2 and MMP9 activity are increased in chronic silica exposure during granuloma formation and markedly upregulated after acute silica exposure. A). qPCR of MMP12 in whole lungs in chronic silicosis across the time course, and 14 d post acute silica exposure. B) Western blot analysis of MMP12 in whole lungs. C) Densitometry of Western Blots for MMP12. D) Zymography of BAL fluids to determine MMP2 and MMP9 enzymatic activity; the contrast was inverted. Each lane represents a unique animal in CON and chronic silicosis (4 d, 7 wk, 14 wk and 28 wk post exposure). E). Zymography comparison of BAL fluids from CON, 28 wk post chronic silica exposure and 14 d post acute silica exposure. Each lane represents a unique animal.